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Sensors and Actuators B 191 (2014) 643– 649

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journa l h om epage: www.elsev ier .com/ locat e/snb

io-inspired sensor for insect pheromone analysis based onolyaniline functionalized AFM cantilever sensor

larice Steffensa,b,∗, Alexandra Manzoli a, Juliano E. Oliveirac, Fabio L. Leited,aniel S. Correaa,b, Paulo Sergio P. Herrmanna,b,e,∗

National Nanotechnology Laboratory for Agribusiness (LNNA), Embrapa Instrumentation, P.O. Box 741, 13560-970 São Carlos, SP, BrazilFederal University of São Carlos (UFSCar), Campus São Carlos, 18052-780 São Carlos, SP, BrazilFederal University of Paraiba (UFPB), Campus João Pessoa, 58051-900 João Pessoa, PB, BrazilFederal University of São Carlos (UFSCar), Campus Sorocaba, 18052-780 Sorocaba, SP, BrazilEmbrapa Labex Europe – Germany, Forschungszentrum Jülich, Institute of Bio-Geoscience 2, 52428 Jülich, NWF, Germany

r t i c l e i n f o

rticle history:eceived 3 July 2013eceived in revised form 7 September 2013ccepted 13 October 2013vailable online xxx

eywords:ensor

a b s t r a c t

Bio-inspired materials designed for mimicking nature has gained attention in the last years, owing totheir appealing properties, such as adhesive and self-cleaning properties. However, studies reportingbio-inspired sensors able to detect minimal amounts of pheromones are still scarce. Here we report onthe functionalization of AFM cantilever with polyaniline (PANI), which was used as a sensor to detectpheromone 2-heptanone. This hormone is very significant for honey bees, which release it as a repellentscent marking to avoid enemies and other bees. The functionalization of the sensor was achieved bydepositing a thin film of PANI in the emeraldine state on the cantilever through spin-coating. Infrared

heromoneunctionalization of cantileveronductive polymerolyaniline

spectroscopy showed that the 2-heptanone was adsorbed by PANI film deposited on cantilever surface.The linear response of the coated cantilever sensor regarding 2-heptanone concentration for distinct tem-peratures was evaluated, as well as its mechanical behavior, hysteresis and storage time. The approachproposed here to functionalize AFM cantilever with PANI film to detect 2-heptanone showed a poten-tial methodology for designing sensors able to detect minimal amounts of pheromones and naturalcompounds.

. Introduction

The development of insect colonies is dependent on the com-unication system established between them, and in some cases

he pheromones released by insects display an important role.pecifically for honeybees, pheromone 2-heptanone, secreted fromandibular glands, plays the role of repellent scent marking and

larm compound to avoid enemies and other robber bees [1,2].-Heptanone is not an exclusive hormone used by bees, but it islso known as an aroma constituent in some types of food, such asoquefort cheese, coffee fruits and others [3–5]. Given the signif-

cance of the pheromone 2-heptanone, new strategies to develophemical sensors, able to mimic biological receptors that can detect

inimal amounts of different types of pheromones, are of great

mportance.

∗ Corresponding authors at: National Nanotechnology Laboratory for Agribusi-ess (LNNA), Embrapa Instrumentation, P.O. Box 741, 13560-970 São Carlos, SP,razil. Tel.: +55 5435209000; fax: +55 5435209090.

E-mail addresses: clarices@uricer.edu.br, claristeffens@yahoo.com (C. Steffens).

925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.snb.2013.10.053

© 2013 Elsevier B.V. All rights reserved.

Furthermore, the study of insect pheromones (semiochemi-cals) for controlled release and environmental monitoring are veryimportant, since they can provide alternatives to reduce the use ofinsecticides and to control insect populations. Among the devicesused for monitoring pheromones, the use of electroantennogram(EAG) (sensors developed with insect antennae) technique ispromising [6]. The EAG technique employs the insect antenna as abiosensor for the identification of electroactive molecules, and con-sists in placing the insect antenna between two electrodes througha conductive gel. Through a chemical stimulus caused by ions dif-fusion across the neuronal membrane, changes in the electricalpotential between the antennas can be recorded. Such method-ology has proven to be of high resolution for analyzing complexmixtures extracted from plants or insects. However, this approachpresents some drawbacks, since it requires a continuous humidifiedair flow to avoid dehydration of the antenna. Moreover, the time fortesting is about 20 min, which is long considering the short lifetimeof the device, and it is very difficult to remove and fix the antenna

to the equipment [7].

The miniaturization of chemical sensors is a target that has beenpursued by scientists in the last years, since they find applicationsin quality food control industry, forensic investigation, biosensors

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44 C. Steffens et al. / Sensors and

or medical diagnosis, portable devices for water and air qualityontrol, among others [8–16]. Depending on the materials compo-ition, miniaturization of sensors can enhance their functionalitynd properties. Among several types of materials employed as thesensing component” of chemical sensors, conductive polymers,hich possess a pi-extended bonding system, are an interesting

hoice, owing to their properties such as high sensitivity on the sur-ounding medium, wide range of synthesis routes, thermal stability17,18]. Polyaniline (PANI), for instance, in its several oxidationtates, has found applications as antistatic agent, anticorrosiveoatings, taste sensors, vapor sensor, electroactive membranes, andlectrochromic displays [15,19–23]. One potential approach thatan help the task of miniaturizing sensors is through the func-ionalization of atomic force microscopy (AFM) cantilevers [24],lthough combined efforts are also necessary to reduce the sizef laser sources coupled to commercial AFM equipment. By coatinghe cantilever with a layer of active material such as a conductiveolymer, and exposing it to the analyte under investigation, it isossible to transduce a chemical reaction into a mechanical stim-lus, such as changes in surface stress, mechanical deformation, orass change [25–27]. This approach is able to detect very small

mounts of the analyte under investigation, with a high degree ofccuracy.

The sensor presented in this work is based on the function-lization of AFM cantilever with spin-coated film of PANI in themeraldine oxidation state, through surface polymerization. Theunctionalization was evaluated by Fourier transform infraredFTIR) spectroscopy and the response of the sensor was evalu-ted by measuring the AFM cantilever deflection in the presencef the pheromone 2-heptanone, at several concentrations and dis-inct temperatures. The cyclic mechanical behavior of the coatedantilever was also studied, in addition to its storage time, sensitiv-ty, reversibility, repeatability and hysteresis, which parameters areundamental for designing reliable and accurate sensor for detec-ing pheromones.

. Experimental

.1. Materials

The PANI interfacial polymerization synthesis in emeraldinexidation state was employed due to its ease of synthesis and also tobtain nanofibers. This synthesis occurs at the interface, betweenwo immiscible and liquid phases, each containing a reagent, asescribed by Huang et al. [10]. For the interfacial synthesis, anilinemonomer) was dissolved in an organic solvent (dichloroethane)ith the oxidant agent (ammonium persulfate) in hydrochloric

cid (HCl) (1 mol/L). All the reagents used had analytical grade.A and were used without any further purification. We employedltrapure water presenting resistivity of 18 M� cm−1 (Milli-Q sys-em Millipore Inc.). Then, the oxidant solution was slowly addedo the monomer solution in order to avoid mixing of the phases.he reaction was performed in a sealed chemical glass bottle for

h, and subsequently filtered (filter paper Milipore 25 �m) andashed with methanol and Mili-Q water. The filtrate was dedoped

n ammonium hydroxide (0.1 M NH4OH) 0.1 mol/L by stirring andried for 12 h in a desiccator under vacuum, which yielded the PANIsalt) in its emeraldine oxidation state. The PANI (salt) was sol-bilized in N-methyl pyrrolidinone (NMP) solvent, because PANI

n emeraldine base (dedoped) is completely soluble NMP secondngelopolous et al. [28]. This dedoped solution was used to func-

ionalize the AFM cantilever surface.The silicon microcantilevers were purchased from NT-MDT

ompany, with resonance frequency of 12 (±2) kHz, spring con-tant of 0.03–0.13 N m−1 and dimensions (rectangular shape) of

tors B 191 (2014) 643– 649

350.0 �m-length; 30.0 �m-width and 0.5–1.5 �m thickness. Themicrocantilevers surfaces were cleaned by employing plasma“sputtering” in high vacuum. The argon gas pressure was lowerthan 0.1 mbar and the background pressure was 0.1 mbar. As exper-imental variables, we employed a radio frequency of 40 kHz, powerof 150 W and treatment temperature of 130 ◦C. Subsequently themicrocantilevers were dried in an oven at 50 ◦C for 10 h and storedin vacuum desiccator.

The pheromone 2-heptanone (MM = 114.18 g/mol, den-sity = 0.82 g/cm3 (20 ◦C) and boiling point: 150–152 ◦C) waspurchased from Aldrich and used without further purification.

2.2. Polyaniline-modified cantilever

The AFM cantilever used to detect 2-heptanone pheromone wasfunctionalized with PANI in salt emeraldine oxidation state, dopedwith HCl, thin film by the spin-coating method. For that purpose,3 �L of the dedoped PANI solution was added to the AFM can-tilever surface, which was placed in the spin coater, which wasoperated using a rotation of 500 rpm for 8 s, increased to 1000 rpmfor 15 s, and finally maintained at 3000 rpm for 60 s. Subsequently,cantilevers sensors were dried in a desiccator under vacuum for12 h at room temperature and finally doped with HCl (1 mol/L).

Silicon wafers (antimony doped type) were purchased com-mercially (NT-MDT) and coated with PANI films using the sameexperimental parameters used to functionalize the AFM cantilever,aiming at verifying the influence of the spin-coated PANI on its sur-face roughness. To detect and distinguish the characteristic peaksof PANI and 2-heptanone through FTIR measurements (Nicolet 470Nexus FTIR spectrometer) silicon wafer coated with doped PANIfilms was prepared using the same experimental conditions usedto functionalize the AFM cantilever. In addition, we also collectedthe FTIR spectra of PANI films on silicon wafers in atmosphere con-taining the pheromone 2-heptanone.

The morphology and roughness of PANI films deposited onthe microcantilevers surfaces were analyzed with a Dimension V(Veeco) AFM, using a silicon nitride tip attached to a cantileverwith a spring constant of 42 N m−1 and resonance frequency of285 kHz. All images were obtained in tappingTM mode at a scanrate of 0.001 kHz. The images and RMS (root-mean-square aver-age) roughness were processed with the aid of the Gwydion© 2.1data analysis software. RMS can be described by [29]:

RMS = 1N

[N∑

i=1

Z2i

]1/2

(1)

being N the number of data points and Z the distance from the meansurface level.

The coated and uncoated microcantilever surfaces were char-acterized with a field emission scanning electron microscope(FE-SEM) (JEOL, JSM-6701F), operating at 5 kV for determining thesize, shape and distribution of the PANI films.

2.3. Sensor performance measurements

The performance of the sensor was evaluated by measuring thedeflection of the coated and uncoated (reference) cantilever in anAFM. The deflections were measured in voltage, with resolutionof millivolts (mV), through monitoring the laser beam position,which is focused at the endpoint of the cantilever and reflected

to a four-quadrant photodiode. This signal value was convertedin nanometers after obtaining the system sensitivity. All measure-ments of deflection were performed in triplicate at static mode ofoperation.

C. Steffens et al. / Sensors and Actuators B 191 (2014) 643– 649 645

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Fig. 1. AFM images (2D) of (a) pure silicon wafers (uncoate

The functionalization was done in just one side of the microcan-ilever sensor. The cantilever displacement, caused by 2-heptanonedsorption, is monitored by using a laser apparatus where the bend-ng is induced by an increase in surface stress on the functionalizedide of the cantilever. Although the oscillating mode in dynamicFM can also be used as a sensing plataform for monitoring liq-id samples, as reported by [30], in this work we used the staticode for analyzing volatiles in an atmosphere with very low con-

ent of pheromone concentration, which results have proven thathe AFM functionalized microcantilever provided high sensitivityo pheromone.

The deflection of the functionalized cantilever as a function ofhe pheromone concentration at 10, 20 and 30 ◦C was measured bynserting with a syringe defined amounts (in ppmv, calculated bysing the ideal gas equation) of 2-heptanone in the AFM chamber,ith sequential measurements of the AFM cantilever deflection.fter the deflection measurements, nitrogen was inserted into thehamber to clean it up, in which procedure was repeated for everyet of measurements.

The flux of the gas inside the chamber was 10 mL/min, whichas obtained using a commercial flowmeter (Aalborg). For exper-

ments at temperatures of 10, 20 and 30 ◦C, a heated water-bathNova Ética, Model 521/2D) was used to maintain the chamberemperature constant. The temperature and humidity inside ofhe chamber (9 mL) was monitored with a commercial sensorSensiriumTM).

The cantilever sensing performance for 2-heptanone in the pres-nce of other pheromones, such as linalool and orange oil (vegetalased pheromones), was evaluated by inserting 5 wt% of the twother pheromones in the AFM chamber, after it was stabilized with000 ppmv of 2-heptanone in an atmosphere at 20 ◦C (controlledemperature). The choice of linalool and orange oil to be investi-ated together with 2-heptanone in the sensor performance wasade based on the fact that they are all floral odorants [31–34]. In

his sense, linalool and orange oil they are “interferents” for playinghe same biological effect of 2-heptanone (pheromone) for bees, buts we could observed in our results, they do not interfere greatlyn the sensor performance for 2-heptanone.

The pheromone content was varied from 0 to 1000 ppmv (cal-ulated by the ideal gas equation) by insertion and removal of-heptanone from the AFM chamber. The sensor response as a func-ion of storage time, for periods of 0, 15, 30, 45 and 60 days, wasvaluated by measuring the deflection of the functionalized can-ilever in the presence of 2-heptanone as a function of time (from 0

o 1000 min). Cantilevers sensors were stored in a vacuum desicca-or and before each analysis were doped with HCl 1 M. Before all the

easurements with the cantilevers sensors, the AFM equipmentas stabilized for 2 h to reach a stable baseline.

(b) silicon wafers modified with spin-coated films of PANI.

3. Results and discussion

3.1. Characterization of PANI film and pheromone

The deposition of PANI films by spin-coating on the surface ofthe AFM cantilever can alter considerably its surface roughness,which was corroborated by the RMS values when comparing thecoated and uncoated surface. Uncoated Silicon surface presented aroughness of 0.002 (±0.0003) �m, while the modified silicon sur-face with PANI film presented a roughness of 0.079 (±0.007) �m,considering a superficial area of 100.0 �m2. The increase in rough-ness shown in Fig. 1(b) is caused by the presence of spin-coatedfilm of PANI over the substrate, which provides chemical sensingproperties to the AFM cantilever. The PANI-coated microcantilever(Fig. 1(b)) displays a uniform layer formed with the presence ofpolymeric aggregates, which plays an important role on the sensorsensitivity.

SEM images of uncoated and PANI-coated microcantilever (seeprocedure in Section 2) are displayed in Fig. 2(a)–(c), in whichfunctionalization followed the same procedure of the silicon sub-strates. Fig. 2(b) and (c) shows in detail the morphology of PANIfilms deposited on the microcantilever tip surface, which displaysnanofibers-like structures, with diameters lower than 100 nm.Such increase in surface area, due to the nanofibers presence, ishighly desirable for designing sensitive layer in sensors, once itprovides a more efficient interaction with the analyte, increas-ing the sensor sensitivity for detecting small changes in analytes[21,35].

To verify the interaction of 2-heptanone with PANI films, weobtained the FTIR spectra of pure PANI film deposited on siliconwafer, and that in an atmosphere containing 2-heptanona. Bothspectra are displayed in Fig. 3. The bands located at 1100, 3240and 3400 cm−1 are typical of doped PANI, and indicate that thedoping state was not change by the presence of the pheromone.Although the two spectra are similar, the interaction of pheromonewith the PANI film deposited on the cantilever shows peak inten-sities at 1640 and 2930 cm−1. The peak at 2930 cm−1 is typical ofC-H stretching, while the peak at 1640 cm−1 is related to carbonylgroups and saturated aliphatic ketone [36,37]. In a previous work,Moore [38], isolated and characterized by FTIR the pheromonereleased by termites, and found high intensity peaks located at3070 and 1640 cm−1, which are close to the ones found here for2-heptanone. This hormone presents a weak physical interactionwith PANI, leading to its adsorption on the PANI functionalized

microcantilever surface. Such interaction do not change the oxi-dation levels of conducting polymers, but alter the properties ofthe sensing materials, which makes this volatile detectable by thesensor.

646 C. Steffens et al. / Sensors and Actuators B 191 (2014) 643– 649

Fig. 2. Scanning electron microscopy (SEM) images (scale bars are displayed in each imamicrocantilever, (b) functionalized with PANI and (c) a close view of the structure formedlike-structure.

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response difference in distinct cycles may be due to saturation on

ig. 3. FTIR spectra of PANI film and PANI film in an atmosphere containing 2-eptanone. The inset shows the chemical structure of 2-heptanone pheromone.

. Sensor performance

The deflection of the coated microcantilever in the presence of000 ppmv of 2-heptanone pheromone as a function of time is dis-layed in Fig. 4. The solid line is a guide to the eye to evidencehe response time of the sensor, given by the experimental data

f deflection (solid squares) as a function of time. The error barsepresent the standard deviation obtained by the triplicate mea-urements. The coated cantilever keeps a deflection of 600 nm in an

ig. 4. Deflection of the coated cantilever with PANI as a function of time in theresence of 1000 ppm of 2-heptanone pheromone. The solid line is a guide to theye to evidence the sensor response time.

ge) of the uncoated and coated microcantilever with PANI: (a) non-functionalized by the spin-coated film of PANI deposited on the cantilever, showing the nanofiber

atmosphere free of 2-heptanone (from 0 up to 175 s). After insertionof 1000 ppmv of 2-heptanone (though an orifice which was 1.5 cmfar from microcantilever, in controlled atmospheric pressure), anddiffusion into the AFM chamber, the cantilever deflection starts todrop off, reaching nearly zero after 250 s. Such behavior evidencesthe interaction between the pheromone adsorbed and the PANI filmdeposited on the AFM cantilever tip. The pheromone 2-heptanonemay have weak physical interactions with the sensing coating,involving absorbing or swelling the polymer. Thus, a change occursin the polymer matrix, causing a swelling of the sensitive coat-ing (on one side of the functionalized cantilever), and thereforeleading to a surface stress during the adsorption of pheromonemolecules. Such adsorption causes the static bending responsiblefor the deflection.

The cyclic mechanical deflection of cantilever as a function oftime was also studied and is displayed in Fig. 5. The cyclic deflectionof the AFM cantilever, achieved by inserting 500 and 1000 ppmv of2-heptanone into the AFM chamber and subsequent cleaning withN2 gas (baseline for a 100 nm deflection), shows an amplitude ofnearly 300 nm and 600 nm, respectively, and presents virtually thesame deflection behavior up to 4000 s, indicating high repeatability.This is a desirable aspect for designing sensor devices. The recoveryand response time for the functionalized cantilever is nearly 120 s.It was also possible to observe that the coated cantilever sensorhas good reversibility of 94 ± 4% during the successive cycles. The

the polymer-based-sensor response of (caused by a doping inter-action). Such saturation may cause a delay in the sensor response

Fig. 5. Mechanical behavior of the coated cantilever with PANI in the presenceof 1000 ppm and 500 ppm of 2-heptanone pheromone, for cyclic deflection as afunction of time.

C. Steffens et al. / Sensors and Actuators B 191 (2014) 643– 649 647

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ig. 6. Bio-inspired sensor properties for the pheromone 2-heptanone, which showshe cantilever deflection as a function of pheromone concentration, measured inhree distinct temperatures.

hen it is exposed to sequential gas insertion cycles. However, it isbserved high baseline stability after successive cycles.

Giving the importance of characterizing the sensor responseegarding the analyte concentration, we measured the AFM func-ionalized cantilever deflection as a function of 2-heptanoneoncentration, at three different temperatures: 10, 20 and 30 ◦C.he results obtained, in the concentration range from 10 up to000 ppmv (Fig. 6), which shows a linear behavior between deflec-ion and pheromone concentration for fixed temperatures. Thisinear dependence was observed for 10, 20 and 30 ◦C, and suchehavior is very helpful for sensing applications, once it allowso predict, for a given temperature, the amount of pheromone inhe environment, through deflection measurements of the coatedantilever. In addition, an increase of pheromone sensitivity as aunction of temperature can also be observed.

Table 1 shows that the pheromone evaporation rate increasedith temperature raise, which was probed by the PANI-

unctionalized microcantilever, while the uncoated microcan-ilever (reference) was not sensitive to the hormone at the set ofemperatures tested (Fig. 6). Such results are a proof that the func-ionalization of the microcantilever with PANI is responsible forroviding sensitivity regarding 2-heptanone hormone. We can also

nfer that there is a linear relation between the sensor responsesor distinct sets of temperature, which is important for practicalpplications. The curves presented in Fig. 6 yielded limit of detec-ion (ppmv) of 31 (10 and 20 ◦C) and 56 (30 ◦C), and sensitivitynm/ppmv) of 0.2 ± 0.01 (10 ◦C), 0.7 ± 0.04 (20 ◦C) and 0.7 ± 0.0330 ◦C). The uncoated microcantilever did not present response forhe investigated pheromone. Marfaing et al. [39] studied the effectf concentration and nature of pheromones in bees by EAG, obtain-ng limit of detection values of tenths of ppm, which are similar tohe ones determined for the PANI-modified cantilever.

The cantilever sensor response in the presence of other

heromones than heptanone was also studied and can be seen

n Fig. 7. For that purpose, we injected into the AFM chamber000 ppmv of 2-heptanone, and after 250 s and 370 s, we injected

able 1ensor properties for 2-heptanone, which shows the cantilever deflection as a func-ion of pheromone concentration, measured in three distinct temperatures.

Temperature (◦C) R2 Sensitivity (nm/ppmv) Detection limit(ppmv)

10 0.99 0.19 ± 0.01 3120 0.99 0.67 ± 0.04 3130 0.99 0.69 ± 0.03 56Reference (20 ◦C) 0.99 0.008 ± 0.001 0

Fig. 7. Functionalized cantilever sensor response for 2-heptanone in the presenceof linalool and orange oil (both are other pheromones) as a function of time.

5 wt% of linalool and orange oil, respectively, which are two flo-ral volatiles routinely present in beekeeping environments. Weobserve that for an empty chamber the maximum deflection ofthe functionalized microcantilever reaches 45 nm. After insertionof 2-heptanone, the deflection was drastically increased to nearly500 nm (increase of more than 1000%), indicating the high sensi-tivity of the functionalized microcantilever to 2-heptanone. Afterinsertion of linalool, the deflection reached nearly 530 nm (increaseof 6% compared to heptanone). Subsequently, we added orangeoil, and the total deflection reached 560 nm (the two volatilesincreased the deflection in 12% when compared to 2-heptanone).The results show that the functionalized cantilever sensor responsefor 2-heptanone, in terms of deflection, is not heavily affected bythe presence of 5% (weight/weight) concentration of the other twovolatiles, indicating the sensing robustness of the functionalizedcantilever.We measured the deflection of the functionalized can-tilever in the presence of 2-heptanone as a function of time for 0, 15,30, 45 and 60 days. The results obtained displayed in Table 2, pointout that the sensor deflection, for the same time range (1000 min),decreases gradually as the storage days increase, i.e., the sensorsensitivity decreases with time (Fig. 8). Usually, the electrical con-ductivity of polyaniline not is stable for long time due to leachingof the dopant, where the dopant molecules HCl can dissolve in theresidual water and subsequently be lost by evaporation [40]. Thisfact can be related to the slight reduction of the sensors sensitiv-ity with the time. According to Bai et al. [41] the performances ofsensors based on conducting polymers are expected to decreasewhen they are stored in air for a relatively long time. This phe-nomenon can be explained as de-doping of conducting polymers.In our case the cantilevers sensors were stored in a vacuum des-iccator and before each analysis were doped with HCl 1 M, whichhelped to keep the good electrical properties of the sensors for 60days. However, even after 60 storage days, the sensor still presents

a reasonable sensitivity of 0.53 ± 0.01 nm/ppmv, but in a lowerlevel compared to 0, 15, 30, days (see Table 2).Comparing the can-tilevers sensors with EAG, it can be observed a large increase in the

Table 2Storage time, detection limit and sensitivity of functionalized cantilever deflectionas a function of time, measured for 0, 15, 30, 45 and 60 days.

Storage time Detection limit (ppmv) Sensitivity (nm/ppmv)

0 31 0.67 ± 0.0415 27 0.69 ± 0.0430 14 0.54 ± 0.0445 15 0.51 ± 0.0460 21 0.53 ± 0.01

648 C. Steffens et al. / Sensors and Actua

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ig. 8. Functionalized cantilever deflection as a function of time, measured for 0,5, 30, 45 and 60 days, showing its storage time behavior.

urability of these cantilevers sensors compared with sensors andiosensors prepared with live parts of the insect for instance, Parkt al. [42,43] reported lifetimes of typical EAG sensors lower than

h, which is much shorter than the ones obtained in this work.

. Conclusions

A sensor based on the functionalization of AFM cantilever withANI film was obtained and used to detect and quantify theheromone 2-heptanone. Infrared spectroscopy spectra showedhat the pheromone 2-heptanone was adsorbed on the PANI filmeposited on the cantilever tip, indicating a chemical affinityetween both compounds. We observed that the functionalizedantilever sensor displayed a linear response on the 2-heptanoneoncentration for distinct temperatures, with good mechanicalehavior for cyclic mechanical deflection. Besides, the sensingroperties of the functionalized cantilever are not greatly affectedy two other volatile pheromones tested, and only a small hystere-is on the mechanical deflection of the cantilever was observed,onfirming its robustness. The sensor storage time, evaluated upo 60 days, showed that the sensor maintains its sensing capac-ty, with low loss of properties. The results described here, basedn a methodology to functionalize AFM cantilever with polyanilineor detecting 2-heptanone, open new opportunities for designing

iniaturized systems used for pheromone analysis and sensing.

cknowledgments

The authors would like to thank Embrapa Instrumentation,esponsible for the National Nanotechnology Laboratory forgribusiness (LNNA) for the infrastructure and facilities, FAPESP

2009/08244-0) and INCT-NAMITEC (CNPq 573738/2008-4) for thenancial support.

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iographies

larice Steffens received her PhD in biotechnology in 2012 from Federal Univer-ity of São Carlos, Brazil. She is currently working a full professor at Department

tors B 191 (2014) 643– 649 649

of Food Engineering, URI – Erechim. His research includes conducting polymers,microcantilever, atomic force microscopy, gas/odor sensing, electronic nose andnanotechnologies.

Alexandra Manzoli received her PhD in science (analytical chemistry) in 2003 fromFederal University of São Carlos, Brazil. She is currently working as a visiting researchat Embrapa Agricultural Instrumentation (Embrapa/CNPDIA), Brazil. Her field ofinterests includes electrochemical quartz crystal nanobalance, semiconductors,atomic force microscopy, conducting polymer, gas/odor sensing, electronic nose andbiosensor.

Juliano E. Oliveira received his PhD degree in materials science and engineeringfrom Federal University of Sao Carlos (DEMa-UFSCar), São Carlos, Brazil in 2011.He is currently Professor at Federal University of Paraiba (DEMat-UFPB), Brazil. Hisresearch interests include polymeric nanostructures and their applications in thebiosystems.

Fabio L. Leite received his MSc and PhD degree in materials science and engineeringfrom the University of São Paulo (USP – São Carlos), in 2002 and 2006, respectively.Currently, he is an Adjunct Professor and Researcher in the Federal University of SãoCarlos (UFSCar), in Sorocaba-SP, Brazil. His research interests are mainly related toatomic force microscopy, nanomedicine, nanoneurobiophysics and nervous systemdiseases.

Daniel S. Correa received his PhD in materials science and engineering in2009 from Universidade de São Paulo, São Carlos, Brazil. During 2009–2010,he worked as a post-doctoral fellow at the Instituto de Física de São Car-los at Universidade de São Paulo. Currently, he is a researcher at EmbrapaInstrumentac ão. His research interest includes nanostructured chemical sensors,optical properties of organic materials and laser microfabrication of polymericmaterials.

nanotechnology.