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2106 IEEE SENSORS JOURNAL, VOL. 15, NO. 4, APRIL 2015
A Nanobiosensor Based on4-Hydroxyphenylpyruvate Dioxygenase
Enzyme for Mesotrione DetectionPmela Soto Garcia, Alberto Lus
Dario Moreau, Jssica Cristiane Magalhes Ierich, Ana Carolina Araujo
Vig,
Akemi Martins Higa, Guedmiller S. Oliveira, Fbio Camargo
Abdalla, Moema Hausen, and Fbio L. Leite
Abstract The herbicide residue from intensive
agriculturalactivity provokes environmental disturbances and human
healthinjuries. Among the enzymatic disruptor herbicides,
mesotrioneis able to inhibit 4-hydroxyphenylpyruvate dioxygenase
(HPPD),which plays a key role in the carotenoid synthesis.
Therefore,enzyme-based sensors are innovative options for
monitoringherbicides used in agriculture. Compared to the standard
sensors,biosensors have assorted advantages, such as practicality,
quickresponse, low cost, and high sensitivity. A nanobiosensor
wasdeveloped herein based on HPPD for mesotrione
detection.Theoretically, the molecular docking and molecular
dynamicssimulation estimated the interacting regions of HPPD
withmesotrione. Experimentally, the atomic force microscope tip
func-tionalization with HPPD immobilized in self-assembled
mono-layers was confirmed by fluorescence microscopy and
atomicforce spectroscopy. The cross-linker
N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride was
responsible for properlypreserving the enzyme on the tip. The
nanobiosensor proposedhere was successfully able to detect
mesotrione molecules. Sucheffectiveness in the development of
nanobiosensors promisesreliable, precise, and low-cost techniques,
which apply to a broadrange of issues, from ecology to
medicine.
Index Terms AFM, AFS, chemical functionalization,nanobiosensors,
molecular docking, molecular dynamicssimulation, mesotrione,
4-hydroxyphenylpyruvate dioxygenase.
Manuscript received June 3, 2014; revised September 23, 2014
andOctober 17, 2014; accepted November 2, 2014. Date of
publicationNovember 20, 2014; date of current version January 29,
2015. Thiswork was supported in part by CNPq (CNPq/INCT,
573742/2008-1), in partby FAPESP (FAPESP/INCT, 2008/57859-5,
2007/05089-9, 2010/00463-2,2010/04599-6, 2013/09746-5,
2013/21958-8, 2011/17840-6, 2014/12082-4),in part by nBioNet, and
in part by CAPES (PNPD/20131505). The associateeditor coordinating
the review of this paper and approving it for publicationwas Dr.
Chang-Soo Kim.
P. S. Garcia, J. C. M. Ierich, A. C. A. Vig, A. M. Higa,G. S.
Oliveira, M. Hausen, and F. L. Leite are with the
NanoneurobiophysicsResearch Group, Department of Physics, Chemistry
and Mathematics,Federal University of So Carlos, So Carlos
18052-780, Brazil (e-mail:[email protected];
[email protected]; [email protected];
[email protected]; [email protected];[email protected];
[email protected]).
A. L. D. Moreau is with the Department of Physics, Federal
Institute ofEducation, Science and Technology of Itapetininga,
Itapetininga 18202-000,Brazil, and also with the
Nanoneurobiophysics Research Group, Departmentof Physics, Chemistry
and Mathematics, Federal University of So Carlos,So Carlos
18052-780, Brazil (e-mail: [email protected]).
F. C. Abdalla is with the Laboratory of Structural and
Functional Biology,Department of Biology, Federal University of So
Carlos, So Carlos18052-780, Brazil (e-mail:
[email protected]).
Color versions of one or more of the figures in this paper are
availableonline at http://ieeexplore.ieee.org.
Digital Object Identifier 10.1109/JSEN.2014.2371773
I. INTRODUCTION
SENSORS are devices used for detection and measurementof
physical properties [1]. There are many different sortsof sensors,
and the understanding of their mechanisms requiresa
multidisciplinary knowledge [2]. When biomolecules areused to
measure relevant biological material, the sensoris defined as a
biosensor [3], [4]. Those biomolecules areusually immobilized
through chemical or physical transducers,creating a surface that
makes possible the direct measurementof a specific molecule [5],
[6]. If the biosensor functions onthe nanometric scale, it is
classified as nanobiosensor [7].
A nanobiosensor is obtained by the functionalization ofAtomic
Force Microscope (AFM) tips and canmeasure forcesat an atomic scale
[8], [9]; the application of those tipson the development of
nanobiosensors is known as AtomicForce Spectroscopy (AFS) [10]. To
build a functionalizedAFM tip requires the chemical modification of
its surface.Such procedure provides three essential benefits: (i) a
highsensitivity device; (ii) detections at the molecular level;
and(iii) simulation of a mimetic microenvironment [11], [12].
The functionalization process requires a previous study ofthe
set of molecules involved, while the availability of theactive site
and substrate orientation are important parametersfor the AFM
measurements [11], [13], [14]. In this context,computer simulation
is an easy and economical approach toestimate intermolecular
interactions. Therefore, the combina-tion of theoretical and
experimental methodologies providesboth macro and micro scale
perspectives to the experiments.
Herbicides act on specific metabolic pathways in plants,as
inhibitors of the synthesis of carotenoids and amino acids.This
triggers photosynthesis failure, which leads to plantstarvation and
death [15]. Herbicides have a deep impact onhuman health and the
environment, and the monitoring of suchagrochemicals in order to
minimize their impact and invitethe development of alternative weed
control procedures inintensive agriculture is crucial. Plants and
animals can sharetargets of homoplasic molecules, and the
metabolization ofherbicide molecules can generate secondary
metabolics. Thesecan be more harmful than the herbicide itself, in
addition tothe surfactant products that compose the herbicide
formula,which have been shown to be extremely toxic to animals
byingestion, inhalation or contact [16]. Among assorted
herbicidetypes, few act by inhibiting specific enzymes. This type
of
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for more information.
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GARCIA et al.: NANOBIOSENSOR BASED ON 4-HPPD ENZYME 2107
Fig. 1. Conversion of HPP to homogentisate, catalyzed by HPPD
enzyme(depicted on green elipse) at the tyrosine pathway [adapted
from literature 21].
inhibition process is a reaction between a molecule and
anenzyme, which forms a complex that blocks the enzymefunction
[16], [17].
The tyrosine catabolism is a common process inplants and animals
and requires key enzymes, such as4-Hydroxyphenylpyruvate
dioxygenase (HPPD) that catalyzeHydroxyphenylpyruvate (HPP) to
homogentisate (Fig. 1),which participate in the energetic
metabolism [18][20].In animals, the lack of such conversion can
adversely affectdifferent metabolic pathways and cause severe
metabolicdisorders, such as Tyrosinemia and Hawkisinuria [18].In
plants, the inhibition of this sequence of metabolic eventsleads to
lower levels of chlorophyll and carotenoids. Thelatter protect the
chlorophyll from excess light. Becauseof those inhibitory events
and the fact that weeds growmuch more quickly than the economic
plant of interest, theherbicide causes leaves of the plant to
blanch and, in a coupleof weeks, causes all photosynthesis to cease
[21].
Several herbicides promote HPPD inhibition. Among them,the
triketone herbicide mesotrione (C14H13NO7S) is usedon pre- and
post-emergency crops. After application, it isabsorbed by leaves
and roots and is readily translocated tothe plant vascular system
[22][24]. The HPPD inhibitors areconsidered low risk
pesticides.
Therefore, to contribute to advances in this researchfield, a
nanobiosensor based on AFM functionalized tipswas developed using
the techniques of AFS and AFM [3],[7], [11], [25] to perform
mesotrione detection through theinhibition of the target enzyme,
HPPD. In our research,the development of nanobiosensors focus on
the study ofneurodegenerative diseases and on agrochemical
detection,especially enzymatic inhibitor herbicides [15],
[26][29].
Mesotrione identification by an AFM tip
nanobiosensorfunctionalized with HPPD, studied hereunder
controlledparameters, can improve the application of
biotechnologyto environmental safety. All techniques and
methodologiesdeveloped in this paper can be adapted to detect
otherpotential contaminants, such as pesticides and trace
metalsthat can harm the environment.
II. MATERIALS AND METHODSA. Molecular Docking Parameters
The initial crystallography structure of the HPPD wasobtained
from the online repository Protein Data Bank (PDB),ID: 3ISQ [30].
The PDB file was prepared for the dock-ing calculations. All
missing residues were completed, andthe molecular structure was
energy-minimized. Eight Mole-cular Dockings were used to determine
the HPPD affinityregions with mesotrione by means of the AutoDock
Tools,version 1.5.6 [31]. The grid boxes had two different sizes,
inAngstrom (): grid boxes 1, 3, 5 and 7 had dimensions ofx = 56, y
= 56 and z = 126, and grid boxes 2, 4, 6 and8 dimensions were x =
68, y = 66 and z = 66, following theprotocol used by Franca et al.
[32]. This last group was anattempt of creating smaller boxes and
obtaining more preciseresults. The Lamarckian Genetic Algorithm
(LGA) was usedto fit the mesotrione molecule in the HPPD, which was
setas a rigid structure, in a total of 100 runs for each
docking.The program was also set to calculate the internal
electrostaticenergy. The RMSD Cluster Tolerance was adjusted to a
limitof 2.0 .
B. Molecular Dynamics SimulationFrom the eight Molecular
Dockings obtained previously,
four systems were chosen based on their final scored energiesand
cluster conformations on which to perform MolecularDynamics (MD).
Nanoscale Molecular Dynamics NAMDversion 2.9 [33] and Visual
Molecular Dynamics VMDversion 1.9.1 [34] were used to prepare the
systems understudy. The main goal was to rearrange the atoms to
minimizeand equilibrate the molecule energetically. The first step
wasto balance the negative charge of the system. The final
chargewas 9.66340 e6 C. The next step was the minimizationof the
systems energies conducted in 100000 steps, total-ing 200 ps. The
temperature adjustments were performed inthe NVT ensemble, at 298 K
and 1.0 atm, using a Langevinthermostat. The cut-off distance of 16
was the sameused inOliveira et al. [35]. The equilibration of the
systems followedthe minimization, with certain modified parameters
in the NpTensemble. Pressure and temperature were controlled using
theLangevin method at 1 atm and 310 K. The cut-off distanceof 12
was needed for accurate interaction contacts over thetrajectory.
The electrostatic interactions were calculated withthe Particle
Mesh Ewald (PME).
C. Experimental Procedure MaterialsAccording to Orry and
Abagyan, (2012) [36], the
homology protocol established a minimum of 25%
sequencesimilarity between two molecules. In this case, the
similaritybetween human and plant HPPD is 33% as performed onthe
BLAST online platform [37]. Therefore, our modelpresents a moderate
similarity that is feasible for use inthe mesotrione herbicide
detection process. In addition,only the pure human HPPD enzyme was
available forpurchase, while the plant one is commercially
restricted.Consequently, in order to evaluate the
nanobiosensorbehavior, the study was conducted using the human
enzyme.
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2108 IEEE SENSORS JOURNAL, VOL. 15, NO. 4, APRIL 2015
The pure human HPPD enzyme was acquired from AcrisAntibodies,
Inc. (San Diego, CA), NCBI NP_001165464. Theenzyme was received
lyophilized, and prior to experiments,it was reconstituted in
Mili-Q water. The mesotrioneagrochemical was purchased from Chem
Service (WestChester, PA) and diluted in pure-grade acetone,
purchasedfrom Qhemis, Hexis Cientfica S/A (Indaiatuba, SP).
Thefollowing pure-grade materials were purchased from Sigma-Aldrich
(St. Louis, MO): monobasic sodium phosphate,dibasic sodium
phosphate, sodium chloride, triethy-lamine (TEA),
3-aminopropyltriethoxysilane (APTES),
andN-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochlo-ride
(EDC). The glutaraldehyde (GLU) (as a 25% aqueoussolution) was
purchased from Nuclear, CAQ (Diadema, SP),and the Casein (CAS) was
obtained from skimmed milkpowder. The fluorochrome used was
fluorescamine(C17H10O4; Sigma-Aldrich, USA). The AFM tips used
weretriangular silicon nitride tips from
NanoWorld-InnovativeTechnologies (Switzerland), model PNP-TR-20.
The cantileverused had the following specifications: overall
thickness:600 nm, length: 200 m, width: 2 28 m, resonancefrequency:
17 kHz, spring constant (k): 0.08 N/m, radius:
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GARCIA et al.: NANOBIOSENSOR BASED ON 4-HPPD ENZYME 2109
Fig. 2. (a) Herbicide-enzyme interaction; (b) enzyme being
injected on thesystem; (c) enzyme-enzyme interaction and (d)
enzyme-free spaces wherethere is no interaction with the herbicide
molecules over the mica substrate.
Fig. 3. Four mesotrione molecules (in green) docked to the
differentregions on the HPPD molecular structure in order to run MD
simulation.Two cofactors are presented, Sodium and Chloride ions
(in black and purple,respectively). The total energies (E values in
kJ.mol1) are represented foreach mesotrione position. The inset
above shows structural formula of themesotrione.
III. RESULTS AND DISCUSSION
A. System EnergyThe immobilization and stability of biomolecular
systems
on functionalized AFM tips is one concern in the design
ofsensitive and selective biosensors [7], [15], [29]. As
mentionedbefore, HPPD (PDB code: 3ISQ) [30] was chosen to act asa
biologic sensor. To evaluate its behavior in an aqueoussolution,
computational simulations (Molecular Docking andMolecular Dynamics
- MD simulation) were performed tomonitor the HPPD fluctuations
such as its interaction energieswith mesotrione herbicide. The most
favorable dockedpositions scored are shown in Fig. 3. Four
different confor-mations of the mesotrione on binding regions of
the HPPDwere considered. By running MD simulations, the systems
TABLE IINHIBITION COEFFICIENT (Ki) AND REFERENCE RMSD, AND
TOTALENERGIES (ET) FOR EACH SYSTEM OF THE MOLECULAR DOCKING
CALCULATIONS AFTER 5 ns OF MD SIMULATION
were energy minimized and energy equilibrated, in order
toanalyze the fluctuations and mobility of the mesotrione-HPPDset
in aqueous solution. The electrostatic and van der
Waalsinteractions were estimated and calculated. The results after5
ns of MD simulation and the average energies are listed inTable
I.
Energetically, the most interactive region of the HPPDenzyme was
found in system 4, but this result cannot bedirectly related to
experimental ones because the orientationof the HPPD on the tip
must be considered, which depends onthe arrangement of multiple
enzymes together. The goal of thiscomputational analysis is to
provide an atomistic perspectiveon the binding regions of HPPD with
mesotrione.
The parameters of inhibition coefficient (Ki) and root
meansquare deviation (RMSD) were analyzed using MolecularDocking
calculations for scoring HPPD binding sites withmesotrione.
According to Franca et al. [32], the concentrationof the herbicide
required to inhibit an enzyme activity isexpected to be lower for
the most favorable binding region.In Table I, mesotrione has the
lowest inhibition coefficient andfavorable interaction energies for
systems 5 and 8. For thesesystems, the adhesion force for
mesotrione was higher. Theanalysis of the Ki revealed that the
inhibition coefficient washigh for systems 1 and 2; as a result,
the clusters formed bythese systems were not suitable for biosensor
requirements.Additionally, the RMSD results revealed a large value
forsystems 1, 2 and 5 (5.99 , 4.16 and 3.37 , respectively)and a
low value for system 8 (1.71 ). Therefore, system 8 ismore
interactive than the other ones, and the experimentalforce curve
obtained with the AFM is strong when the HPPDis oriented to the
substrate similar to system 8.
B. Root Mean Square Deviation Protein StabilityFig. 4 shows the
computed RMSD for the 4 evaluated
systems. The HPPD structural fluctuations were monitoredduring 5
ns of MD simulation in the presence of mesotrionemolecules. As
shown, all systems have similar averageRMSD: 1.5 . The fluctuations
can be attributed to the saltbridges and hydrogen bonds formed and
broken over time.According to Franca et al. [32], charged amino
acids such asARG, LYS, ASP and GLU located on the border can
inducenew hydrogen bonds between water molecules and HPPD,causing
small structural fluctuations. The amino acids that
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2110 IEEE SENSORS JOURNAL, VOL. 15, NO. 4, APRIL 2015
Fig. 4. Structural fluctuation of the HPPD at the presence of
mesotrioneherbicide during 5 ns.
TABLE IINUMBER OF SALT BRIDGES AND HYDROGEN BONDS FORMED
AFTER
5 ns OF MD SIMULATION
contribute more hydrogen bonds were ASP, GLU and LYS,while CYS,
ILE and TRP do not have relevant contributionsto the total number
of hydrogen bonds during the MDsimulation. Moreover, during all
simulations, salt bridgenumbers from the HPPD were almost the same,
and HPPDstructure was preserved. As shown in Table II, the numberof
salt bridges remained constant during the MD simulation;thus, the
enzymatic structure was not affected by the solvent.As a result, no
effect was observed at the loops and sidechains, which have high
RMSD values, and no denaturationwas detected on the HPPD enzyme
structure.
Table II shows the number of salt bridges and hydrogenbonds at
the beginning (0 to 2.5 ns) and at the end(2.5 to 5.0 ns) of the
simulation. Both initial and late patternspresented similarities,
which consequently revealed that theposition of the mesotrione has
no influence on the HPPDenzyme structure. Finally, the
computational results showedthat HPPD is stable enough to be used
as a biosensor andhas specific interactive regions to
mesotrione.
C. Support of Tip Functionalization byFluorescent Labeling
According to literature [39], fluorescence microscopy (FM)is
usedto confirm the AFM tip functionalization. Thecombination of AFM
and other techniques, such as confocallaser scanning microscopy and
fluorescent imaging, provides abetter understanding of biological
studies, enlarging the possi-bilities of investigation and giving
more detailed information.
Fig. 5. The AFM tips observed by two microscopy techniques:
brightfield (a, c) and fluorescent mode (b, d). The same tip is
observed inboth techniques for the nonfunctionalized-control group
(a, b) and for thefunctionalized one (c, d). The tips
functionalized with HPPD and conjugatedwith fluorescamine
(Si/HPPD-F) presented intense blue fluorescence while
thenonfunctionalized ones remained dark in fluorescent mode (b).
This qualitativeresult confirmed the HPPD presence on tips.
TABLE IIIADHESION FORCE (AF) OBTAINED WITH AFS EXPERIMENTS
FOR
CONTROL (600 ADHESION FORCE CYCLES)
They may become important tools in medicine, detectingdiseases
in early stages [40]. In this paper, the use of FMto detect HPPD
confirmed the AFM tip functionalization.The images obtained by FM
showed that the methodologywas effective in attaching the
biomolecule to the tip (Fig. 5).Furthermore, other studies [41]
suggest functionalizationevaluation by confocal microscopy and
mediated by indirectfluorescent labeling to be an effective tool to
scan and detectall labeling distribution on the tip surface at
higher resolution.
D. Mesotrione Detection by AFM Tip NanobiosensorThe first
experimental data were obtained from control
tips, organized as follows: (type 1) clean tips, without
anyfunctionalization; (type 2) tips functionalized with APTES
andTEA; (type 3) tips functionalized with APTES, TEA, EDCand CAS.
These three control tips were used to perform forcemeasurements in
the AFM liquid cell, over the sample withthe herbicide. The
obtained adhesion force data were lowerthan expected, at values
around 0.4 nN for measurements insolution [42], [43].
The force measurement characterizations of all control tipswere
used as parameters for the nanobiosensor according tothe values
shown in Table III. The type 2 tips showed highadhesion values,
most likely due to the interaction between the
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GARCIA et al.: NANOBIOSENSOR BASED ON 4-HPPD ENZYME 2111
Fig. 6. Representative histograms for the sets of measurements
to theNanobiosensor (AFnb) and System Inhibition (AFsi). The
adjustment wasperformed by Gauss curve.
APTES and the herbicide. Compared to those with APTES, thetype 3
tips showed lower values because CAS favors the inhi-bition of the
active sites of APTES, as recently reported [44].Limanskii [45]
also performed a functionalization on siliconnitride AFM probes,
using APTES vapors. In that work, theuse of the linking agent
Disuccinimidyl suberate (DSS) wasfollowed by albumin attachment.
The model proposed byLimanskii [45] and the one presented here
share the use ofAPTES vapors to successfully induce modifications
on the tip.
The nanobiosensor, developed through the functionaliza-tion of
the HPPD enzyme on APTES, TEA, EDC andCAS, is expected to present a
higher value of adhesioncompared to the control tests, specifically
detecting the her-bicide mesotrione. The results presented here are
in agree-ment with our previous experiments using
nanobiosensorswith diclofop, atrazine and metsulfuron-methyl
agrochem-icals [15], [26], [28]. The high adhesion values that
wereobtained confirm the effectiveness of the
functionalizationmethod under aqueous conditions. Therefore, the
protocolestablished for the nanobiosensors is straightforward and
canbe applied to assorted detections.
After obtaining the control data, the nanobiosensors
weredeveloped and tested. First, a functionalization
methodologywith APTES, TEA, GLU, and HPPD was tested (data
notshown). However, this functionalization was not efficient anddid
not provide good adhesion force values, which led to theconclusion
that the biomolecule HPPD did not link properlyon GLU, and the
active sites were most likely not in favorablepositions to link to
the substrate.
Fig. 6 shows that the adhesion values ranged approximately1.5 nN
and reached a recover of 63% to the nanobiosensorand 35% to the
system inhibition. These values are includedin Table IV. Although
the frequency changed, the adhesionforce remained the same, as
expected. According to theproposed model, the system was probably
inhibited due toHPPD linkage on mesotrione (Fig. 2d).
The method was evaluated by measuring the nanobiosensorforce
value, which was two times higher than with thecontrol tips. This
finding implies that the HPPD was properly
TABLE IVADHESION FORCE (AF) OBTAINED WITH AFS EXPERIMENTS
FOR
NANOBIOSENSOR AND INHIBITION
orientated on the tip due to the EDC cross-linker,
probablyexposing the interaction sites to mesotrione molecules.
All data sets presented demonstrate that the
nanobiosensordeveloped here was effective for mesotrione detection.
Theinhibition parameter is very informative as it verified
thefidelity by the characterizing approach, while the FM
directlyconfirmed the functionalization. The promising
resultsobtained by our research group [7], [15], [29], [46]
bringforward insights to the study of intermolecular
detections.
IV. CONCLUSIONThe combination of theoretical and experimental
studies
identified possible regions where the herbicide
mesotrioneinteracts on the HPPD molecular structure.
Additionally,the AFM adhesion measurements showed the accuracy
ofthe functionalized HPPD nanobiosensor, which was alsocorroborated
by the FM tip labeling. The next step of ourinvestigation is to
compare the results from human HPPD withthe plant HPPD because the
latter is more directly affected bymesotrione molecules. Finally,
the originality of the biosensorproposed in this paper is based on
AFS categorical detectionof herbicides for environmental
monitoring.
ACKNOWLEDGMENTSThe authors of this paper would like to thank
M. Castilho de Almeida Moura for the tip drawingsusing Corel
Draw. They acknowledge the Post-GraduationProgram of Biotechnology
and Environment Monitoring ofthe Federal University of So Carlos,
Sorocaba.
REFERENCES[1] J. Riu, A. Maroto, and F. X. Rius, Nanosensors in
environmental
analysis, Talanta, vol. 69, no. 2, pp. 288301, Apr. 2006.[2] J.
R. Stetter, W. R. Penrose, and S. Yao, Sensors, chemical
sensors,
electrochemical sensors, and ECS, J. Electrochem. Soc., vol.
150, no. 2,pp. S11S16, Feb. 2003.
[3] D. K. Deda et al., Atomic force microscopy-based molecular
recogni-tion: A promising alternative to environmental contaminants
detection,in Current Microscopy Contributions to Advances in
Science and Tech-nology, vol. 5, A. Mndez-Vilas, Ed., 1st ed.
Badajoz, Spain: FormatexResearch Center, 2012, pp. 130.
[4] A. F. Melo, Desenvolvimento preliminar de um biossensor
enzimticopara determinao de taninos hidrolisveis, Univ. Fed. Rio de
Janeiro,Rio de Janeiro, Brazil, Tech. Rep., 2008.
[5] A. Berquand et al., Antigen binding forces of single
antilysozyme Fvfragments explored by atomic force microscopy,
Langmuir, vol. 21,no. 12, pp. 55175523, Jun. 2005.
[6] O. H. Willemsen, M. M. Snel, A. Cambi, J. Greve, B. G. De
Grooth,and C. G. Figdor, Biomolecular interactions measured by
atomic forcemicroscopy, Biophys. J., vol. 79, no. 6, pp. 32673281,
Dec. 2000.
[7] D. K. Deda et al., The use of functionalized AFM tips as
molecularsensors in the detection of pesticides, Mater. Res., vol.
16, no. 3,pp. 683687, Jun. 2013.
-
2112 IEEE SENSORS JOURNAL, VOL. 15, NO. 4, APRIL 2015
[8] C. Steffens, F. L. Leite, C. C. Bueno, A. Manzoli, and P. S.
Herrmann,Atomic force microscopy as a tool applied to
nano/biosensors, Sensors,vol. 12, no. 6, pp. 82788300, 2012.
[9] P. Hinterdorfer and Y. F. Dufrne, Detection and localization
of singlemolecular recognition events using atomic force
microscopy, NatureMethods, vol. 3, no. 5, pp. 347355, May 2006.
[10] F. Leite, Theoretical models for surface forces and
adhesion and theirmeasurement using atomic force microscopy, Int.
J. Molecular Sci.,vol. 13, no. 12, pp. 1277312856, 2012.
[11] F. L. Leite and P. S. P. Herrmann, Application of atomic
force spec-troscopy (AFS) to studies of adhesion phenomena: A
review, J. Adhes.Sci. Technol., vol. 19, nos. 35, pp. 365405, Jan.
2005.
[12] A. Noy, D. V. Vezenov, and C. M. Lieber, Chemical force
microscopy,Annu. Rev. Mater. Sci., vol. 27, no. 1, pp. 381421,
1997.
[13] B. Dordi, J. P. Pickering, H. Schnherr, and G. J. Vancso,
Invertedchemical force microscopy: Following interfacial reactions
on thenanometer scale, Eur. Polym. J., vol. 40, no. 5, pp. 939947,
May 2004.
[14] K. Wadu-Mesthrige, B. Pati, W. M. McClain, and G.-Y. Liu,
Disaggre-gation of tobacco mosaic virus by bovine serum albumin,
Langmuir,vol. 12, no. 14, pp. 35113515, Jan. 1996.
[15] A. C. N. da Silva et al., Nanobiosensors based on
chemically modifiedAFM probes: A useful tool for metsulfuron-methyl
detection, Sensors,vol. 13, no. 2, pp. 14771489, Jan. 2013.
[16] J. E. Casida and M. Tomizawa, Insecticide interactions with
-aminobutyric acid and nicotinic receptors: Predictive aspects
ofstructural models, J. Pesticde Sci., vol. 33, no. 1, pp. 48,
2008.
[17] N. Nugaeva, K. Y. Gfeller, N. Backmann, H. P. Lang, M.
Dggelin, andM. Hegner, Micromechanical cantilever array sensors for
selective fun-gal immobilization and fast growth detection,
Biosensors Bioelectron.,vol. 21, no. 6, pp. 849856, Dec. 2005.
[18] G. R. Moran, 4-hydroxyphenylpyruvate dioxygenase,
ArchivesBiochem. Biophys., vol. 433, no. 1, pp. 117128, Jan.
2005.
[19] M. Kavana and G. R. Moran, Interaction of
(4-hydroxyphenyl)pyruvatedioxygenase with the specific inhibitor
2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione,
Biochemistry, vol. 42,no. 34, pp. 1023810245, Sep. 2003.
[20] S. Molchanov and A. Gryff-Keller, Inhibition of
4-hydroxyphenylpyruvate dioxygenase by
2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione, Acta
Biochim.Polonica, vol. 56, no. 3, pp. 447454, 2009.
[21] K. Grossmann and T. Ehrhardt, On the mechanism of action
andselectivity of the corn herbicide topramezone: A new inhibitor
of4-hydroxyphenylpyruvate dioxygenase, Pest Manage. Sci., vol.
63,no. 5, pp. 429439, May 2007.
[22] R. Martinazzo, D. P. Dick, M. M. Hirsch, S. B. Leite, andM.
do Carmo Ruaro Peralba, Sorption of atrazine and mesotrionein
oxisols and estimation of contamination potential, Qumica Nova,vol.
34, no. 8, pp. 13781384, Jan. 2011.
[23] P. H. Raven, Biologia Vegetal, vol. 9, G. Koogan, Ed., 6th
ed., Rio deJaneiro, Brazil: Guanabara Koogan, 2001.
[24] G. Meazza et al., The inhibitory activity of natural
products on plantp-hydroxyphenylpyruvate dioxygenase,
Phytochemistry, vol. 60, no. 3,pp. 281288, Jun. 2002.
[25] G. Binnig, C. F. Quate, and C. Gerber, Atomic force
microscope, Phys.Rev. Lett., vol. 56, no. 9, pp. 930933, Mar.
1986.
[26] B. B. Souza et al., Modern trends in nanobiosensors using
atomicforce microscopy, presented at the Latin Amer. Conf.
Metastable andNanostruct. Mater., So Carlos, Brazil, 2012.
[27] E. F. Franca, A. M. Amarante, and F. L. Leite, Introduction
to atomicforce microscopy simulation, in Science, Technology,
Applicationsand Education, vol. 4, A. Mndez-Vilas, Ed., 1st ed.
Badajoz, Spain:Formatex Research Center, 2010, pp. 13381349.
[28] C. C. Bueno, Desenvolvimento de um nanobiossensor para o
moni-toramento da qualidade ambiental no setor agrcola, M.S.
thesis, Dept.Phys. Chem. Math., Univ. Federal So Carlos, So Paulo,
Brazil, 2013.
[29] C. C. Bueno et al., Nanobiosensor for diclofop detection
based onchemically modified AFM probes, IEEE Sensors J., vol. 14,
no. 5,pp. 14671475, May 2014.
[30] E. S. Pilka et al., Crystal structure of human
4-hydroxyphenylpyruvatedioxygenase, Biochemistry, vol. 43, no. 32,
pp. 1041410423, 2004.
[31] G. M. Morris et al., AutoDock4 and AutoDockTools4:
Automateddocking with selective receptor flexibility, J. Comput.
Chem., vol. 30,no. 16, pp. 27852791, Dec. 2009.
[32] E. F. Franca, Designing an enzyme-based nanobiosensor using
molec-ular modeling techniques, Phys. Chem. Chem. Phys., vol. 13,
no. 19,pp. 88948899, 2011.
[33] L. Kal et al., NAMD2: Greater scalability for parallel
moleculardynamics, J. Comput. Phys., vol. 151, no. 1, pp. 283312,
1999.
[34] W. Humphrey, A. Dalke, and K. Schulten, VMD: Visual
moleculardynamics, J. Molecular Graph., vol. 14, no. 1, pp. 3338,
Feb. 1996.
[35] G. S. Oliveira, Molecular modeling of enzyme attachment on
AFMprobes, J. Molecular Graph. Model., vol. 45, pp. 128136, Sep.
2013.
[36] A. J. W. Orry and R. Abagyan, Homology ModelingMethods
andProtocols, vol. 857, 1st ed. New York, NY, USA: Humana Press,
2012.
[37] Basic Local Alignment Search Tool. [Online].
Available:http://blast.ncbi.nlm.nih.gov
[38] A. L. D. Moreau and M. A. Cotta. (Nov. 17,
2005).Processamento e Funcionalizao de Pontas Para
AplicaesBiologicas de Microscopia de Fora Atomica. [Online].
Available:http://www.bibliotecadigital.unicamp.br/document/?code=vtls000385613,accessed
Sep. 17, 2014.
[39] G. S. Lorite et al., Surface physicochemical properties at
the micro andnano length scales: Role on bacterial adhesion and
Xylella fastidiosabiofilm development, PLoS One, vol. 8, no. 9, p.
e75247, Sep. 2013.
[40] B. J. Haupt, A. E. Pelling, and M. A. Horton, Integrated
confocaland scanning probe microscopy for biomedical research, Sci.
World J.,vol. 6, pp. 16091618, Dec. 2006.
[41] A. Li et al., Molecular mechanistic insights into the
endothelialreceptor mediated cytoadherence of plasmodium
falciparum-infectederythrocytes, PLoS One, vol. 6, no. 3, p.
e16929, Mar. 2011.
[42] U. Dammer et al., Specific antigen/antibody interactions
measured byforce microscopy, Biophys. J., vol. 70, no. 5, pp.
24372441, May 1996.
[43] R. De Paris, T. Strunz, K. Oroszlan, H.-J. Gntherodt, and
M. Hegner,Force spectroscopy and dynamics of the biotin-avidin bond
stud-ied by scanning force microscopy, Single Molecules, vol. 1,
no. 4,pp. 285290, Dec. 2000.
[44] M. Breitenstein, R. Hlzel, and F. F. Bier, Immobilization
of differentbiomolecules by atomic force microscopy, J.
Nanobiotechnol., vol. 8,no. 1, p. 10, May 2010.
[45] A. P. Limanskii, Functionalization of amino-modified probes
for atomicforce microscopy, Biophysics, vol. 51, no. 2, pp. 186195,
Apr. 2006.
[46] A. C. N. da Silva et al., Nanobiosensors exploiting
specific interactionsbetween an enzyme and herbicides in atomic
force spectroscopy,J. Nanosci. Nanotechnol., vol. 14, no. 9, pp.
66786684, Sep. 2014.
Pmela Soto Garcia was born in Sorocaba, Brazil,in 1984. From
2006 to 2007, she performed under-graduate research at the
Biomonitoring Laboratory,Faculty of Technology at Sorocaba
(FATEC-SO),Sorocaba. In 2008, at her second undergraduateresearch
at the Dante Pazzanese Institute, So Paulo,Brazil, she studied
devices for medical applications.She received the B.S. degree in
health technologyfrom FATEC-SO, in 2008. In 2009, she
studiedmicrobiology in public health at the Adolfo LutzInstitute,
So Paulo. She received the M.Sc. degree
in biotechnology and environmental monitoring from the Federal
Universityof So Carlos (UFScar), Sorocaba, in 2014. Since 2012, she
has developedatomic force microscopy (AFM) tips nanobiosensors, and
is a specialist inAFM and Nanotechnology with the
Nanoneurobiophysics Research Group,UFScar, where she is currently
pursuing the Ph.D. degree in nanobiosensors.
Alberto Lus Dario Moreau was born in So Paulo,Brazil, in 1977.
He received the B.S. degree inphysics, and the M.Sc. and Ph.D.
degrees from theState University of Campinas, Campinas, Brazil,
in2003, 2005, and 2011, respectively. He is currentlya Professor
and Coordinator of the Basic PhysicsLaboratory at the Federal
Institute of Education,Science and Technology, Itapetininga,
Brazil. Since2013, he has been with the
NanoneurobiophysichsResearch Group, Federal University of So
Carlos,Sorocaba, and has experience in biophysics with an
emphasis on functionalization and immobilization of biomaterials
surfaces andinterfaces, force spectroscopy with atomic force
microscopy (AFM), AFMtopographic analysis of biomaterials, carbon
nanotubes and graphene, andbiosensors in semiconductor
platforms.
-
GARCIA et al.: NANOBIOSENSOR BASED ON 4-HPPD ENZYME 2113
Jssica Cristiane Magalhes Ierich was born inSorocaba, Brazil, in
1991. She received the degree intechnology on biomedical systems
from the Facultyof Technology at Sorocaba, Sorocaba, in 2011,
andthe M.S. degree in biotechnology and environmentalmonitoring
from the Federal University of So Car-los (UFSCar), Sorocaba, in
2014. In 2012, she hadthe opportunity to study 3-D structures of
proteinsusing homology modeling and molecular dynam-ics simulation.
Also, she has studied enzymaticinhibition process by herbicides for
nanobiosensors
applications. She is currently pursuing the bachelors degree in
biologicalsciences and the Ph.D. degree at UFSCar. Her Ph.D. study
is focused on thedescription of antigen-antibody interaction by
means of computational andtheoretical approaches.
Ana Carolina Araujo Vig was born in So Paulo,Brazil, in 1992.
She is currently pursuing the bach-elors degree in chemistry at the
Federal Universityof So Carlos (UFSCar), Sorocaba, Brazil. In
2010,she began teaching Chemistry for private students,preparatory
courses at Corporative University, andtutoring at Aprendiz
Reinforcement School. She alsotutored the students of UFSCar
coursing Physics Iin 2012. In 2011, she joined the GNN
ResearchGroup, functionalizing atomic force microscopy tipsfor the
study of nanobiosensors. Currently, she has a
Scientific Initiation in Theoretical and Computational
Chemistry, studying theIgG antibody, specially its binding site,
and its relation to multiple sclerosis.
Akemi Martins Higa is currently pursuing thebachelors degree in
biological sciences from theFederal University of So Carlos
(UFSCar), Soro-caba, Brazil. She was born in So Paulo, Brazil,
in1992. She joined the GNN Research Group in 2012,studying the
immobilization of enzymes on atomicforce microscopy tips. Since
2013, she has studiedthe development of quantum dots
functionalizationtechniques to cover them with biomolecules, suchas
antibodies and antigens. The main purpose of herstudies with the
group is to develop a nanobiosensor
that promotes an accurate and early diagnosis for multiple
sclerosis disease.
Guedmiller S. Oliveira received the B.S. and M.S.degrees in
physical chemistry from the Federal Uni-versity of Uberlndia,
Uberlndia, Brazil, in 2006and 2009, respectively, and the Ph.D.
degree inphysical chemistry from the Federal University ofSo Carlos
(UFSCar), Sorocaba, Brazil, in 2013.Since 2007, he has worked with
computer simulationproviding an atomistic point of view for
experi-mental procedures. His expertise lies on quantummechanics
theory, molecular dynamics simulation,and it combines results from
experimental and theo-
retical analysis through statistical thermodynamics to improve
comprehensionof the macromolecular phenomena. He currently holds a
post-doctoral positionwith UFSCar.
Fbio Camargo Abdalla received the bachelorsdegree in biological
sciences from So Paulo StateUniversity, Rio Claro, Brazil, in 1996,
the mas-ters degree in biological sciences with a minor inmolecular
cellular biology from So Paulo StateUniversity and the University
of Utrecht, Utrecht,The Netherlands, in 1999, the Ph.D. degree in
bio-logical sciences with a minor in molecular cellularbiology from
So Paulo State University and KeeleUniversity, Keele, U.K., in
2002, and the Post-Doctoral degree from So Paulo State University,
in
2006. He is currently a Professor with the Federal University of
So Carlos,Sorocaba, Brazil. He has experience in cell and molecular
biology with anemphasis on structural and functional biology and
chemical ecology.
Moema Hausen was born in Rio de Janeiro, Brazil,in 1977. She
received the Ph.D. degree from theState University of Rio de
Janeiro, Rio de Janeiro,in 2009. After three years performing her
firstpost-doctoral assistance at the Brazilian Center forPhysics
Research, Rio de Janeiro, she is currentlyinvolved in a second one,
at the Biotechnologyand Environmental Monitoring Post-Graduation
Pro-gram, Federal University of So Carlos, Sorocaba,Brazil. In
2000, she started in biomedical scientificlaboring in the following
themescell biology, his-
tology, protozoology, transmission, and assorted
state-of-the-art microscopytechniques, such as the scanning
electron, transmission electron, fluorescence,and confocal ones.
Her main goals actually are the application of high-endmicroscopy
techniques to integrated approaches on materials and
biologicalsciences.
Fbio L. Leite was born in Itanhaem, Brazil.He received the B.Sc.
degree in physics from SoPaulo State University, Rio Claro, Brazil,
in 2000,and the M.Sc. and Ph.D. degrees in materials scienceand
engineering from the University of So Paulo,So Carlos, Brazil, in
2002 and 2006, respec-tively. From 2007 to 2008, he was a
Post-DoctoralResearcher with the Alan Graham MacDiarmidInstitute of
Innovation and Business, Embrapa Agri-cultural Instrumentation
(Embrapa), So Carlos,with Dr. O. N. de Oliveira, Jr., Dr. L. H. C.
Mattoso
(Embrapa), and A. G. MacDiarmid, and was a recipient of the
University ofPennsylvania Nobel Prize in Chemistry in 2000. His
efforts at the MacDiarmidInstitute focused on conducting polymers,
nanosensors, and atomic forcemicroscopy (AFM) with environmental
applications. Since 2009, he has beenan Assistant Professor and a
Researcher with the Federal University ofSo Carlos, Sorocaba,
Brazil, and the Head of the NanoneurobiophysicsResearch Group. He
has authored over 50 published papers, five books,10 book chapters,
and holds two patents. His research interests are relatedto the
development of nanobiosensors using AFM and computational
nano-technological for application in the studies of a variety
neurodegenerative andautoimmune diseases.
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