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Electrochimica Acta 87 (2013) 717– 723

Contents lists available at SciVerse ScienceDirect

Electrochimica Acta

jou rn al hom epa ge: www.elsev ier .com/ locate /e lec tac ta

he use of mixed self-assembled monolayers as a strategy to improve thefficiency of carbamate detection in environmental monitoring

uliana Cancinoa,∗, Claudia A. Razzinob, Valtencir Zucolottoa, Sergio A.S. Machadob

Nanomedicine and Nanotoxicology Laboratory, Physics Institute of São Carlos, University of São Paulo, CP 369, 13560-970 São Carlos, SP, BrazilChemistry Institute of São Carlos, University of São Paulo, CP 780, 13560-970 São Carlos, SP, Brazil

r t i c l e i n f o

rticle history:eceived 23 April 2012eceived in revised form1 September 2012ccepted 15 September 2012vailable online xxx

eywords:ixed self-assembled monolayers

cetylcholinesteraseiosensorarbaryl

a b s t r a c t

We report a simple strategy to obtain an efficient enzymatic bioelectrochemical device, in which theenzyme acetylcholinesterase (AChE) was immobilized on gold electrodes functionalized with mixedself-assembled monolayer (SAMmix) of 11-mercaptoundecanoic acid (11-MUA) and 2-mercaptoethanol(C2OH). Development of the modified electrodes included the chemical adsorption of SAMmix on goldsurface followed by immersion in AChE solution, resulting in the final Au/SAMmix/AChE configuration.For comparison, the electrochemical performance of Au/11-MUA/AChE and Au/C2OH/AChE electrodeswere also investigated. The performance of the modified electrodes toward acetylthiocholine hydroly-sis was investigated via cyclic voltammetry and chronoamperometric measurements, revealing a fastincrease in anodic current with a well-defined peak upon addition of acetylthiocholine iodide to theelectrolytic solution. The anodic currents for Au/SAMmix/AChE electrodes were significantly higher thanthose for Au/11-MUA/AChE and Au/C2OH/AChE electrodes. The Au/SAMmix/AChE architecture providedby the SAMmix surface promoted a high oxidation current of thiocholine at 0.31 V without the need of

electron mediators. The chronoamperometric biosensor developed here provided a linear response tocarbaryl in the concentration range of 0 to 1.75 �M. The detection limit and quantification values forcarbaryl were found to be 3.45 × 10−10 M and 1.15 × 10−9 M, respectively. Michaelis–Menten kinetics,Kapp

M , of 0.46 mM was obtained, indicating that the electrode architecture employed is advantageous forfabrication of enzymatic devices via physical adsorption process with improvement of the biocatalyticproperties.

. Introduction

Carbamate pesticides refer to a specific class of organophos-hates pesticides, involved in a number of physiological processes.espite the importance regarding the environmental monitor-

ng of carbamates, the efficient detection of such molecules stillepresents a challenge from the analytical point of view. Theain toxic effects of carbaryl(1-naphthyl-N-methylcarbamate), for

xample, include the inhibition of the catalytic activity of acetyl-holinesterase (AChE), an enzyme that catalyzes the hydrolysis ofhe acetylcholine neurotransmitter [1,2].

Due to the potential risk of pesticide residues to human health,he use of pesticides is strictly regulated. Many countries haveeveloped programmes for pesticides monitoring, including the

∗ Corresponding author at: Laboratório de Nanomedicina e Nanotoxicologia,rupo de Biofísica Molecular, Instituto de Física de São Carlos, Universidade de Sãoaulo, Avenida Sãocarlense, 400, CP 369, CEP: 13560-970 São Carlos, São Paulo,razil. Tel.: +55 16 3373 9875; fax: +55 16 3371 5381.

E-mail addresses: jucancino@yahoo.com.br, jcancino@ursa.ifsc.usp.brJ. Cancino).

013-4686/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.electacta.2012.09.080

© 2012 Elsevier Ltd. All rights reserved.

U.S. Environmental Protection Agency (EPA) that established theuse of HPLC with a postcolumn derivatization and fluorescence fordetection of carbaryl. Although this method is sensitive and wellestablished, its regular use remains complex and expensive instru-mentation is required [3,4]. Electrochemical methods, including theuse of amperometric biosensors, on the other hand, are simple,inexpensive and highly sensitive methods [5,6]. Biosensors basedupon immobilized AChE, in particular, are based on the inhibitionof AChE by the binding of pesticides molecules to the active sitesof the enzyme. The latter devices have shown high sensitivity andexcellent behavior in pesticides detection [7–9].

Strategies related to the efficient immobilization of enzymes,including AChE, in transductor surfaces represent an importantresearch topic in bioelectroanalysis, especially when improved,highly sensitivity is required [10–13]. In order to assure the high-est possible efficiency of biological devices, enzymes have beenimmobilized on different transducers for a wide range of applica-

tions [14–20]. Some techniques for enzyme immobilization on solidsurfaces, including adsorption, covalent cross-linking and entrap-ment in various materials, usually uses glutaraldehyde or othercross-linking agents to covalently connect the transducer to the

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nzymes. The main disadvantage in the use of cross-linking agentss that they often block both the electrode surface and the enzymesctive sites, lowering the electroactivity of the transducer andhe biocatalytic activity of the enzymes [21,22]. As an alterna-ive, reports have been made on the use of mixed self-assembled

onolayers (SAMmix), i.e., SAMs containing two different species oflkanethiols with different carbon chains length, as a biocompat-ble matrix to immobilize enzyme [23,24]. Such strategy exhibitsttractive properties, including excellent film-forming ability,ood adhesion, high permeability, biocompatibility, mechanicaltrength, susceptibility to chemical modifications and electronransfer properties [23,25]. It is important to note that despite theirxtensive application in several research fields [26–31], the appli-ation of mixed SAMs free from cross-linking agents in biosensorss an alternative strategy to protein immobilization is relativelyew.

The use of a SAMmix is one of the most attractive methodologieso promote different surface arrangements on electrode surfaces,epresenting an efficient strategy to understand the direct electronransfer from the enzyme to surface without needing of media-ors or cross-linking agents. The association of different propertiesf alkanethiols with distinctive carbon chain lengths allows theormation of special surface structures composed by short chainslands surrounded by long chain insulating phase [32]. It is possi-le to optimize the electron and mass transfer through the islandshile the long chain alkanethiols act as support for enzymes immo-

ilization.An efficient biosensor response is determined by the enzyme

D structure which in turn is influenced by the physicochemicalharacteristics of the enzyme. According to Sussman et al. [33–35]he enzyme dipole moment might serve to attract the positivelyharged substrate of AChE into and down the active-site. In general,he aromatic surface of the active-site might serve as a weak affinityolumn down which the substrate could skip or move towards thective site via electrostatic interactions. Shafferman et al. [36,37]howed that amino acid residues around the entrance to the active-ite made a major contribution to the dipole moment. Alternatively,he opposite side of AChE molecule consists in the positive pole ofhe dipole and, as a consequence, may attract anions.

Here, we propose the use of mixed SAMs to develop a biosensoror carbaryl based in the inhibition of immobilized AChE by physi-al adsorption process [34,38]. The SAMmix exhibit an architectureormed by a self-assembled monolayer (SAM) of mixed alkanethiolsith short (2-mercaptoethanol) and long (11-mercaptoundecanoic

cid) chains. The shorter chain alkanethiols provide the electronnd mass transfer through the islands while the long insulatinglkanethiols support enzyme structure [32]. The idea is to retain theaximum enzymatic activity without using cross-linking agents orediators. This strategy is possible because of the deprotonation

f the interfacial carboxylic groups from 11-mercaptoundecanoiccid at basic pH, as well as the subsequent binding interaction, asydrogen bonding and van der Waals interactions, between theegatively charged interface and the positive pole of the enzyme39,40]. Acetylcholinesterase was used as an enzyme model forheir special physicochemical characteristics.

. Experimental

.1. Chemicals

All chemicals were of analytical grade. Acetylcholinesterase

E.C. 3.1.1.7, type VI-S, 425.94 U mg−1) from Electrophoruslectricus (electric eel), acetylthiocholine iodide (ATI), 11-ercaptoundecanoic acid (11-MUA) and 2-mercaptoethanol

C2OH) were purchased from Sigma–Aldrich and used without

Acta 87 (2013) 717– 723

purification. Carbaryl (1-naphthyl-N-methylcarbamate) (99%purity) was obtained from Bayer. Ethanol (99%, Merck) was usedas received. Ultrapure water (18.2 M� cm) produced by MilliporeMilli-Q system was used to prepare all the aqueous solutionsemployed here.

2.2. Electrochemical set-up

A gold disk electrode (2 mm diameter) embedded in polyte-trafluoroethylene (PTFE) was used as the work electrode. Thiselectrode presented a geometric area of 0.030 cm2. A Pt foil(2 cm2 geometric area) and an Ag/AgCl (KCl 3 mol L−1) wereused as auxiliary and reference electrodes, respectively, andwere employed in all experiments, in a borosilicate conventionalthree-electrodes one compartment electrochemical cell with aPTFE cover. Electrochemical experiments were performed using aPGSTAT40 Autolab electrochemical system (Eco Chemie, Utrecht,Netherlands) equipped with the PGSTAT-12 and GPES softwares(Eco Chemie, Utrecht, Netherlands).

2.3. Electrode pre-treatment

First, the gold disc electrode was cleaned in a “piranha” solution,which is a mixture of 30% H2O2 and concentrated H2SO4 (1:3, v/v).Second, the electrode was polished with emery paper grade 2000,followed by polishing in 0.08 �m diamond powder. Then it wasultrasonicated in ethanol solution to remove undesirable particles.Finally, the electrodes were electrochemically cleaned by cyclingthe potential between −0.1 and 1.8 V in 0.5 M H2SO4 at scan ratesof 0.5, 0.1 and 0.05 V s−1. The characteristics voltammograms of aclean gold electrode were obtained after approximately 100 cycles.An electroactive surface area of 0.120 ± 0.011 cm2 was determinedfrom the reduction peak of oxygen adsorption on the gold electrode[41] and has a satisfactory reproducible area even after repeatedusage. This surface treatment was the most appropriate to producea clean and reproducible surface [32,42].

2.4. Preparation of SAMs and SAMmix on gold surface

After rinsing with deionized water and ethanol the electrodewas dried with nitrogen gas and immersed in a 1.0 × 10−3 Methanolic solution of the correspondent alkanethiols for 2 h. Theelectrode was then rinsed with ethanol to remove weakly adsorbedmolecules and immediately used in the electrochemical experi-ments. The modification of Au surface with SAMmix was conductedby immersion of the Au electrode in an ethanolic solution contain-ing a mixture of 1.0 × 10−3 M 11-MUA and 1.2 × 10−4 M C2OH for2 h, where was expected a mean radius value of 3.8 �m of each 11-MUA molecule, accordingly to previous papers [32]. The modifiedelectrode was rinsed with deionized water before each experiment.SAMmix electrodes were characterized as described in previouspapers [32].

2.5. Au/SAMmix/AChE biosensor preparation

AChE was immobilized onto the SAMmix electrode by a phys-ical adsorption process, in which the forces involved are mostlyhydrogen bonds and van der Waals interactions. The latter forcesare sufficient to enable reasonable binding without damage of theenzyme and have been reported in previous papers [43–45]. TheSAMmix electrode was immersed in a solution of the AChE (1 g L−1

in 1 mM phosphate buffer saline (PBS) solution, pH 6.5) for approx-

imately 12 h. When not in use, the Au/SAMmix/AChE biosensor wasstored in the PBS solution. The performance of the immobilizedenzyme was monitored after three measurements by the oxidationcurrent in the chronoamperometric measurements in the presence

imica Acta 87 (2013) 717– 723 719

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J. Cancino et al. / Electroch

f ATI at a fixed concentration. The apparent Michaelis–Mentenonstant (Kapp

M ) was estimated by chronoamperometry in the pres-nce of different ATI concentrations (0.25, 0.50, 1.0, 1.5, 2.0 and.5 mM) at a potential of 0.31 V for 200 s. The Kapp

M value wasbtained upon plotting a graphic of I vs [ATI] and performing non-inear fitting of the data using the software Origin.

.6. Amperometric detection of pesticide

The determination of carbamates by enzymatic biosensors isased on the inhibition rate of enzyme activity by the pesticide.n biosensors using AChE, the enzyme may be inhibited by carba-

ates, in which an ester of thiocholine as the acetylthiocholine issed as substrate. Acetylthiocholine is enzymatically hydrolyzed

nto thiocholine, which is oxidized at constant potential, accordingo the reactions:

CH3)3N+CH2CH2SCOCH3 + H2OAChE−→(CH3)3N+CH2CH2SH

+ CH3COOH (R1)

(CH3)3N+CH2CH2SH → 2H+ + 2e−

+ (CH3)3N+CH2CH2S–SCH2CH2N+(CH3)3 (R2)

Carbaryl determination was performed by the incubationethod. The inhibition rate was calculated using Eq. (1):

(%) = I0 − I1I0

× 100 (1)

here I0 was the current of the Au/SAMmix/AChE biosensor inBS pH 6.5 containing ATI 3.0 × 10−3 M, and I1 was the currentf the Au/SAMmix/AChE biosensor in PBS pH 6.5 containing ATI.0 × 10−3 M after incubation with the pesticide.

Experimentally, the Au/SAMmix/AChE biosensor was firstmmersed in 10 mL of carbaryl solution (PBS pH 6.5) for 10 min atifferent concentrations, obtained from a 10 × 10−3 M stock solu-ion in acetonitrile. After this incubation time, the biosensor wasinsed and placed in the electrochemical cell containing the enzymeubstrates (ATI solution 3.0 × 10−3 M in PBS pH 6.5) for chronoam-erometric measurements.

The Au/SAMmix/AChE biosensor was subjected to chronoam-erometric measurements at a potential of 0.31 V (vs Ag/AgCl)or 200 s. The Au/SAMmix/AChE biosensor was inserted into thelectrochemical cell containing 3.0 × 10−3 M ATI solution preparedirectly in 10 × 10−3 M of PBS pH 6.5 and the current was recordedfter 200 s and set as I0. For the incubation by pesticide solu-ion, the Au/SAMmix/AChE was transferred to the incubation cellt different concentrations for 10 min [46]. After incubation, theu/SAMmix/AChE biosensor was rinsed and immediately inserted

nto electrochemical cell for chronoamperometric measurementsnd the current collected at 200 s was recorded as I1. The elec-rode was regenerated after every measurement. Regeneration ofhe enzymatic activity was fully obtained by keeping the biosen-or, after each inhibition measurement, for 5 min, in PBS pH 6.5ontaining ATI 3.0 × 10−3 M.

To obtain the analytical curve the carbaryl concentration wasaried from 0 to 1.75 × 10−6 M (0, 0.25, 0.75, 1.10, 1.2, 1.5 and.75 × 10−6 M). The detection limit (LD) and quantification (LQ)ere calculated using Eqs. (2) and (3), respectively:

D = 3 × SB

b(2)

Q = 10 × SB

b(3)

mix

(dotted line) and Au/SAMmix/AChE (full line) electrodes in PBS pH 6.5 containing ATI3.0 × 10−3 M. Scan rate: 0.05 V s−1.

where SB is the standard deviation of the blank solution (for 7 dif-ferent experiments) and b the slope of the analytical curve [47].

3. Results and discussion

The electrochemical behavior of the new biosensor was inves-tigated on bare Au, Au/SAMmix and Au/SAMmix/AChE modifiedelectrodes using cyclic voltammetry. Fig. 1 shows the cyclicvoltammetric profiles for the electrodes in PBS pH 6.5 contain-ing ATI 3.0 × 10−3 M. For the Au and the Au/SAMmix surfacesit was observed a pair of peaks at Epa = 0.51 V and Epc = 0.43 V.This reversible electrochemical process is a consequence of oxi-dation process of iodide at the electrode surface [48]. The iodideis present as a counter ion in the enzymatic substrate acetylthio-choline molecule (acetylthiocholine iodide). It is important to notethat acetylthiocholine is not an electroactive species. Experimentsadding sodium iodide in ATI 3.0 × 10−3 M PBS solution pH 6.5 (datanot shown), show the increase in oxidation and reduction peaks forthe iodide.

In addition to the redox process of iodide, an extra anodicirreversible process is observed at 0.31 V in the voltammogramfor the Au/SAMmix/AChE surface. The anodic peak at 0.31 V cor-responds to the oxidation of thiocholine, which is produced duringthe enzymatic reaction between the ATI substrate and the immobi-lized AChE enzyme. This electrochemical signal is quite difficult tobe observed and, as reported in several papers, it requires a medi-ator to be evidenced [49–51]. Here, the SAMmix architecture on theelectrode surface promoted mass and electron transfer reaction,allowing oxidation at 0.31 V, a less positive potential if comparedto other modified electrodes recently reported for the same appli-cation [52,53]. Moreover, the electrochemical behavior of iodidewas split into two anodic and two cathodic peaks, due to inter-actions with oxidized thiocholine also present in the electrodesurface.

Individual monolayers of Au/C2OH/AChE and Au/11-MUA/AChEwere also analyzed towards thiocholine oxidation and thevoltammetric curves are shown in Fig. 2. For comparison, voltam-mograms obtained using different surface architectures are alsoshown.

The peak current observed for the electrode modified with

Au/SAMmix/AChE (�) is approximately 10 times higher thanthat observed for the electrode modified with the short chainthiol, Au/C2OH/AChE (�). The 11-MUA-modified electrode, Au/11-MUA/AChE (�), showed no response in potential window analyzed.

720 J. Cancino et al. / Electrochimica Acta 87 (2013) 717– 723

Fig. 2. Linear sweep voltammograms for Au/C2OH/AChE (�), Au/11-MUA/AChE(�) and Au/SAMmix/AChE (�). Note that oxidation current is 10 times higher onA −1

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Fig. 3. Chronoamperometric currents (black squares) of thiocholine oxidation

measurements as well as the analytical time, an incubation timeof 10 min must be used to provide the best results. On the otherhand, regeneration of the enzymatic activity was fully obtained

Table 1Operational kinetics parameters for immobilized AChE films regarding thechronoamperometric detection of ATI and carbaryl.

Biosensor configuration KappM

(mM)a LD (×10−8 M)b Reference

Immobilization on mixed SAMs 0.46 0.035 This workNanocomposites using

multiwalled carbonnanotube coating goldnanoparticles

0.27 290 [52]

Adsorption intonanostructured PbO2/TiO2/Ti

1.34 0.010 [60]

Entrapment ofacetylcholinesterase inpoly(vinyl alcohol) cryogelmembrane

0.40 230 [61]

Silica sol–gel filmimmobilization onto thecarbon paste electrode

0.45 0.39 [53]

Gate surface of an ion-sensitivefield-effect transistor (ISFET)

2.70 0.10 [62]

u/SAMmix/AChE than on Au/C2OH/AChE. Scan rate: 0.05 V s . Electrolyte: PBS pH.5 containing ATI 3.0 × 10−3 M.

omparing the peak current of the three modified electrodeshowed in Fig. 2, it is observed a good response in terms of catalyticehavior for this new architecture, Au/SAMmix/AChE, which is capa-le of providing a faster pathway for electron and mass transfer and

ncreasing the voltammetric signal, for this application. In addition,o electrochemical signal from enzymatic activity was observed

or Au/11-MUA/AChE electrode, revealing that electron and massransfer were totally blocked by this insulating layer. Therefore, its possible to propose the new platform as an excellent transducer,acilitating the electron and mass transfer and increasing the cur-ent when compared to a single alkanethiol monolayer that usedross-linking and mediators agents [54].

To understand the affinity of the immobilized AChE to theAMmix surface, we study the electrochemical response of theu/SAMmix/AChE to ATI using chronoamperometry. When differentTI concentration was added to the buffer solution, the oxidationurrent increased to reach a steady state value and at higher ATIoncentrations the response showed a shape of Michaelis–Mentenrocess. The Michaelis–Menten behavior for the proposed biosen-or was verified by chronoamperometry measurements to 0.31 Vor 200 s. The oxidation potential of thiocholine was determinedy cyclic voltammetry as shown in Fig. 2. The current values of thehronoamperometric curves for several concentrations of acetylth-ocholine in the solution exhibited limit currents controlled by massransport for whole process, including adsorption, dimerizationnd diffusion process, as displayed in Fig. 3.

The fitting of the data suggests the Michaelis–Menten rela-ionship, indicating that an enzymatic process is occurring on thelectrode surface. At high substrate concentration, the reaction ratepproaches constant values, Imax, that can be calculated by nonlin-ar fitting as 4.38 × 10−6 A.

The apparent Michaelis–Menten constant (KappM ) was calculated

or the biosensor Au/SAMmix/AChE, through statistical analysis ofhe nonlinear fit by the software Origin and shown in Fig. 3. This

ichaelis–Menten analysis is based on an assumption that theurrent at 200 s is surely a steady state one and is completely pro-ortional to the enzymatic hydrolysis reaction rate. According toichaelis–Menten equation [55,56], the enzymatic kinetic reaction

an be written as:

1iSS

= 1imax

+ KappM

imax· [ATI] (4)

dependence on ATI concentration using Au/SAMmix/AChE electrode in PBS pH 6.5containing ATI 3.0 × 10−3 M (Michaelis–Menten plot). The black line represents non-linear fit by the software Origin.

where the apparent Michaelis–Menten constant, KappM , defines the

affinity between the enzyme and its substrate. In general, the lowerthe value of Kapp

M , the higher the affinity of the enzyme and itssubstrate, i.e., the higher the biosensor sensitivity [57,58]. Fromthe nonlinear fit, Kapp

M = 0.46 × 10−3 M for the Au/SAMmix/AChEbiosensor. This Kapp

M value is as low as other values presented inthe literature, as shown in Table 1. This Kapp

M value showed that theimmobilized AChE on SAMmix maintained its biocatalytic activityand the modified electrode created a more favorable surface for theattachment of the AChE. These results suggest that the biosensordeveloped with a SAMmix transducer presents an appropriate archi-tecture in which the enzyme exhibits the highest possible catalyticrate. Fig. 4 shows a scheme of such architecture.

Carbaryl pesticides are known to inhibit the reaction betweenAChE and acetylthiocholine, hence they have been widely used asreferences to test acetylcholinesterase biosensors. In this type ofbiosensors, incubation time and reactivation are key parameters[59]. Considering the sensitivity of the chronoamperometric

Carbon paste electrode 1.12 4.2 [63]

a KappM

values are from ATI concentration.b LD values are from carbaryl concentration.

J. Cancino et al. / Electrochimica Acta 87 (2013) 717– 723 721

Fig. 4. Schematic representation of the Au/SAMmix/AChE biosensor electrode. Thescheme represents the modified gold electrode (yellow surface) with SAMmix com-posed by 11-MUA and C2OH molecules (composed by the green line that representsthe carbon chain and blue sphere that represents the SH group) and also by theenzyme AChE molecules (represents by two red spheres) that is adsorbed onto the11-MUA molecules. In this scheme we represent the active process performanceof the modified electrodes toward acetylthiocholine hydrolysis upon addition ofacetylthiocholine iodide (ATI) in a solution and the inhibition process. The violetsphere represents the carbaryl molecule which is absorbed on the active site of theAChE inhibiting the acetylthiocholine access the enzyme active site. (For interpre-tw

b5cnOc

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StledbIbv

btits

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the superiority and efficiency of the use is the SAMmix should be

ation of the references to color in this figure legend, the reader is referred to theeb version of this article.)

y keeping the biosensor, after each inhibition measurement, for min, in PBS pH 6.5 containing ATI 3.0 × 10−3 M. The inhibitionhronoamperometric measurement time of 200 s used here wasot enough to release the carbaryl from the active site of AChE.therwise a constant current value in the chronoamperometricurve had not been observed.

A series of chronoamperometric experiments were carried outpon collecting the response signal of Au/SAMmix/AChE electrodesefore and after incubation in a solution at different pesticide con-entrations. The results are presented in Fig. 5A. The relationshipetween the inhibition percentage (I%) and the carbaryl concentra-ion (ranging from 0 to 1.75 × 10−6 M) is presented in Fig. 5B. One

ay observe that upon increasing the concentration of pesticidehe inhibition increased linearly within the interval of inhibition,etween 12.4% and 97.3%. The linear fitting procedure yielded annalytical curve defined by y = 5.73 × 107x − 3.62, with a correlationoefficient of 0.9678.

The standard deviation for the blank signals was estimated atB = 6.59 × 10−9 A. These data were used to calculate the detec-ion limit of the biosensor as LD = 3.45 × 10−10 M and quantificationimit of LQ = 1.15 × 10−9 M. Notably, this value is lower than thosexhibited by some AChE biosensors described in literature, asepicted in Table 1 [52,53,60–63], excepted by the system reportedy Wei et al. [60], which requires the manipulation of heavy metals.

n comparison to similar electrodes containing multiwalled car-on nanotube-coating gold nanoparticles [52], for example, the LDalues found here are ca. 104 times lower.

A total of 50 measurements were performed in 15 days. Theiosensor was preserved in PBS pH 6.5 and stored in a refrigera-or at 4 ◦C. The response of the biosensor showed maximum value

n the early days, with subsequent decline in value. After 15 dayshe biosensor worked with 75% of their initial performance (nothown).

ent carbaryl concentrations in PBS pH 6.5 containing ATI 3.0 × 10 M. Carbarylconcentrations used: 0, 0.25, 0.75, 1.10, 1.2, 1.5 and 1.75 × 10−6 M. (B) Analyticalcurve.

4. Conclusion

The modification of polycrystalline gold surfaces with mixedself-assembled monolayer composed of short- and long-chain alka-nethiols has been proven to be an efficient strategy to produceelectrochemical transducers for pesticides detection. The long-chain alkanethiol served as the basis to anchorage of enzymemolecules while the short-chain systems facilitating the oxidationof thiocholine. The performance of the biosensor toward carbaryldetermination was very satisfactory, presenting low detectionlimits comparable to as those obtained using nanostructuredPbO2/TiO2/Ti modified surfaces. The main advantages offered withthe use of SAMmix is the absence of cross-linking agents, whichmay hamper enzyme activity, as well as the faster mass transport,provided by the island configuration. On the other hand, physi-cal factors as agitation and flow rate can affect the desorption ofthe enzyme from the support in a physical adsorption process. Theease of preparation of the mixed SAMs-modified surfaces, as wellas the high catalytic activity and the high sensitivity and repro-ducibility exhibited by the Au/SAMmix/AChE point to the possibleapplications of this methodology in the commercial developmentof pesticides biosensors. Nonetheless is important to notice that

verified in future by comparing the results to those for a singlecomponent SAM of short alkyl chain thiol with a terminal carboxylgroup.

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cknowledgements

The authors acknowledge the financial support of the Fundac ãoe Amparo à Pesquisa do Estado de São Paulo – FAPESP (Proc. No.008/00546-5), CNPq and CAPES.

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