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Analytical and Bioanalytical Chemistry DOI 10.1007/s00216-010-3975-2Rapid detection of Aspergillus flavus in rice using biofunctionalized carbon nanotube field effecttransistorsVillamizar Maroto Rius

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Metadata of the article that will be visualized in OnlineFirst

1 Article Title Rapid detection of Aspergillus flavusin rice usingbiofunctionalized carbon nanotube field effect transistors

2 Article Sub- Title3 Article Copyright -Year Springer-Verlag 2010(This will be the copyright line in the final PDF)4 Journal Name Analytical and Bioanalytical Chemistry5

CorrespondingAuthor

Family Name Rius6 Particle7 Given Name F. Xavier8 Suffix9 Organization Universitat Rovira i Virgili

10 Division Department of Analytical and Organic Chemistry11 Address Marcell. Domingo, s/n, Tarragona 43007, Spain12 e-mail [email protected]

Author

Family Name Villamizar14 Particle15 Given Name Raquel A.16 Suffix17 Organization Universitat Rovira i Virgili18 Division Department of Analytical and Organic Chemistry19 Address Marcell. Domingo, s/n, Tarragona 43007, Spain20

Organization

University of Pamplona21 Division Department of Microbiology

22 Address Km 1, Va Bucaramanga, N. de S. Colombia,Pamplona 31080, Spain

23 e-mail24

Author

Family Name Maroto25 Particle26 Given Name Alicia27 Suffix28 Organization Universitat Rovira i Virgili29 Division Department of Analytical and Organic Chemistry30 Address Marcell. Domingo, s/n, Tarragona 43007, Spain31 Organization cole Suprieure de Chimie Organique et Minrale

(ESCOM)32 Division33 Address EA 4297 TIMR, 1 alle du rseau Jean-Marie

Buckmaster, Compigne 60200, France34 e-mail

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35Schedule

Received 3 February 201036 Revised 8 June 201037 Accepted 28 June 201038 Abstract In the present study, we have used carbon nanotube field effect transistors

(FET) that have been functionalized with protein G and IgG to detectAspergillus flavusin contaminated milled rice. The adsorbed protein G on thecarbon nanotubes walls enables the IgG anti-Aspergillusantibodies to be well

oriented and therefore to display full antigen binding capacity for fungalantigens. A solution of Tween 20 and gelatine was used as an effectiveblocking agent to prevent the non-specific binding of the antibodies and othermoulds and also to protect the transducer against the interferences present inthe rice samples. Our FET devices were able to detect at least 10 g/g of A.flavusin only 30 min. To evaluate the selectivity of our biosensors, Fusariumoxysporumand Penicillium chrysogenumwere tested as potential competingmoulds for A. flavus. We have proved that our devices are highly selective toolsfor detecting mycotoxigenic moulds at low concentrations in real samples.

39 Keywordsseparated by ' - ' Biosensor - Field effect transistors - Carbon nanotubes - Moulds - Aspergillusflavus

40 Foot noteinformation

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1

23 ORIGINAL PAPER

4 Rapid detection of Aspergillus flavus in rice

5 using biofunctionalized carbon nanotube field

6 effect transistors

7 Raquel A. Villamizar & Alicia Maroto & F. Xavier Rius

8 Received: 3 February 2010 /Revised: 8 June 2010 /Accepted: 28 June 20109 # Springer-Verlag 2010

10

11 Abstract In the present study, we have used carbon12 nanotube field effect transistors (FET) that have been13 functionalized with protein G and IgG to detectAspergillus14 flavus in contaminated milled rice. The adsorbed protein G15 on the carbon nanotubes walls enables the IgG anti-16 Aspergillus antibodies to be well oriented and therefore to17 display full antigen binding capacity for fungal antigens. A18 solution of Tween 20 and gelatine was used as an effective19 blocking agent to prevent the non-specific binding of the20 antibodies and other moulds and also to protect the21 transducer against the interferences present in the rice22 samples. Our FET devices were able to detect at least23 10 g/g of A. flavus in only 30 min. To evaluate the24 selectivity of our biosensors, Fusarium oxysporum and25 Penicillium chrysogenum were tested as potential competing26 moulds for A. flavus. We have proved that our devices are27 highly selective tools for detecting mycotoxigenic moulds at28 low concentrations in real samples.

29 Keywords Biosensor. Field effect transistors . Carbon30 nanotubes . Moulds . Aspergillus flavus

31Introduction

32 Aspergillus flavus is a mycotoxigenic and filamentous33mould widely distributed in nature on a variety of food34and agricultural products. It causes a wide range of35diseases, ranging from hypersensitive reactions to invasive36infections associated with angioinvasion. A. flavus is after37 Aspergillus fumigatus, the second leading cause of invasive38and non-invasive aspergillosis [1]. In addition, it is one of39the most significant fungi in the spoilage of grain during40storage and is therefore responsible for the economic41devaluation of the grain through mycotoxin contamination42[2]. Rice is one of the most important staple foods for a43large part of the world's human population. It can grow in44different agro-climatic conditions and is vulnerable to45Aspergillus infection in the field as well as in storage.46It is difficult to develop a general method for detecting47all mycotoxins because they are metabolites that display48different chemical structures. A goodapproach, therefore, is to49detect the mycotoxigenic moulds in the early stages of growth50 before they can produce mycotoxins [3]. Conventional51methods for identifying and detecting mycotoxigenic fungi52in rice and foods use cultures in different media or53immunological methods [4]. Viable plate count method uses54specific media which allow the moulds to grow over a55surface. A high mould count indicates possible aflatoxin56contamination [3]. Other methods currently available analyse57the electrical properties of the contaminated food substratum58which are related with the mould grow. Indirect conductance59measurement can be performed and correlated with the60amount of food spoilage yeast [5].61Polymerase chain reaction (PCR)-based methods are62highly specific and have been used to detect aflatoxigenic63strains of A. flavus [6, 7]. Passone et al. [8] developed a64real-time PCR (RT-PCR), directed against the nor-1 gene of

R. A. Villamizar: A. Maroto : F. X. Rius (*)Department of Analytical and Organic Chemistry, UniversitatRovira i Virgili,

Marcell. Domingo, s/n,43007 Tarragona, Spaine-mail: [email protected]

A. Marotocole Suprieure de Chimie Organique et Minrale (ESCOM),EA 4297 TIMR, 1 alle du rseau Jean-Marie Buckmaster,60200 Compigne, France

R. A. VillamizarDepartment of Microbiology, University of Pamplona,Km 1, Va Bucaramanga, N. de S. Colombia,31080 Pamplona, Spain

Anal Bioanal ChemDOI 10.1007/s00216-010-3975-2

JrnlID 216_ArtID 3975_Proof# 1 - 06/07/2010


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65 the aflatoxin biosynthetic pathway, to monitor and66 quantify Aspergillus section Flavi population in peanuts.67 Sensitivity tests demonstrated that the test can detect DNA68 amounts accounting for a single conidium of Aspergillus69 parasiticus. It is a rapid, sensitive and specific assay, not70 inhibited by matrix effects. However, it requires the use of71 a convenient DNA extraction procedure in order to obtain72 reliable results; as a consequence, the method is time73 consuming.74 Immunological methods like enzyme-linked immuno-75 sorbent assay (ELISA) rely on the specific binding of an76 antibody to an antigen. This technique is based on the77 detection of mycelial antigens or extracellular antigens78 secreted by moulds with detection range of 1100 g/mL79 [3]. However, they require the use of specific labels.80 Electronic noses are used to detect volatiles and odours81 produced by the fungi [912]. These results can subsequently82 be correlated with certain parameters such as mycelia83 development on cereal grain or mycotoxin production [9].84 This methodology is able to distinguish between infected85 and non-infected samples and can sometimes even distinguish86 between non-mycotoxigenic and mycotoxigenic fungi. How-87 ever, the methodology is time consuming because the devices88 have to be previously trained to avoid false-positive or false-89 negative results. Moreover, the information obtained is often90 qualitative.91 To overcome this problem, mass and electrochemical92 sensors have been developed to quickly detect and quantify93 fungi [1315]. Nugaeva et al. [14] used protein-modified94 microcantilevers to detect in situ the growth process of95 Aspergillus niger and Saccharomyces cerevisiae. After 4 h96 of incubation, the cantilever detected the presence of the97 fungi and was able to distinguish between alive and dead98 cells. Despite the emerging methods, some performance99 parameters such as sensitivity and selectivity must be100 improved.101 Single-walled carbon nanotubes (SWCNTs) are one-102 dimensional nanostructures that display remarkable phys-103 ical and mechanical properties. Moreover, they can be104 incorporated into a carbon nanotube field effect transistor105 (CNTFET) to make a biosensor with improved perfor-106 mance parameters [1621]. In this study, we used a new107 electrochemical biosensor based on carbon nanotube field108 effect transistors to quickly and selectively detect A. flavus109 in rice samples. In order to improve the sensitivity of our110 devices, we have used protein G; this allows the proper111 orientation of the polyclonal anti-Aspergillus antibodies112 and, therefore, optimal antigen binding. Thus, anti-113 Aspergillus antibodies are able to provide two specific114 binding sites for fungal membrane antigens. A phosphate115 buffer saline (PBS) solution containing Tween 20 and116 gelatine was used as a blocking agent to prevent the non-117 specific binding (NSB) [22] of the antibodies and other

118moulds and also to protect the transducer against the119interferences present in rice samples. The sensor120showed high selectivity in the presence of competing121moulds. Our devices are label free and able to detect12210 g/g of A. flavus with a time response of 30 min. All123these performance parameters are clearly better than those124of the methods currently used to detect mycotoxigenic125fungi.

126Materials and methods

127Proteins and biochemicals

128Anti-Aspergillus (i.e. a polyclonal rabbit anti-Aspergillus;1291 mg/mL) was purchased from Oxford Biotechnology Ltd.130(Oxford, UK). It was dissolved in PBS to a final131concentration of 8 g/mL (pH=7.2) and stored at20 C132until use. Protein G from Streptococcus sp. recombinant,133expressed in Escherichia coli (1 mg/mL), PBS (pH 7.4),134Tween 20 and gelatin from cold-water fish skin were135obtained from Sigma-Aldrich. Sabouraud dextrose agar136(SDA) was provided by oxoid, and brain heart infusion137(BHI) broth and buffered peptone water were obtained from138Scharlau Chemie Microbiology and prepared according to139their specifications.

140Apparatus

141A mini-orbital shaker Stuart was used to prepare the fungal142biomass. An environmental scanning electron microscope143(E-SEM), Quanta 600 (FEI, Hillsboro, OR, USA), was used144to take images of the as-grown networks of SWCNTs and145the functionalization process. Electrical measurements were146taken using a 4157A Agilent semiconductor parameter147analyser and a Wentworth Laboratories MP1008 probe148station.

149Fungal growth and preparation of fungal biomass

150Lyophilized strains of A. flavus CECT 2684, Penicillium151chrysogenum CECT 2307 and Fusarium oxysporum152CECT 2154 were obtained from the Spanish Type Culture153Collection (Valencia, Spain). They were rehydrated with154sterile water, subcultured at least three times on SDA and155incubated at 30 C for 7 days to form single colonies [23].156The spores from the subcultured media were washed from157the surface with a PBS solution. Subsequently, flasks158containing 100 mL of BHI were inoculated with 1 mL of159the spore suspension obtained before and were incubated160at 30 C and agitated at 120 rpm for 7 days [24]. The161cultures were harvested and the mycelium was separated162by filtration through Whatman No. 5 filter paper [25]. The

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163 mycelium was collected, washed with sterile distilled164 water and dried in the oven at 105 C until its weight was165 stable [26]. Then, it was diluted in PBS (w/v) to produce166 the final samples ofA. flavus at 10, 100 and 1,000 ng/mL.167 The same procedure was followed forP. chrysogenum and168 F. oxysporum.

169 Rice sample preparation

170 Milled-rice samples were obtained from Arrosaires del171 Delta de lEbre, Tarragona, Spain. Twenty-five grammes172 of the sample was dissolved in 225 mL of 0.1% sterile173 buffered peptone water and sterilized by autoclaving at174 121 C for 15 min. The rice sample was then shaken and175 diluted in the same solvent for up to 105-fold. After that,176 the resulting samples were artificially contaminated by177 spiking 1 mL of A. flavus at 10, 100 or 1,000 ng/mL in178 9 mL of the diluted rice sample. In this way, the final179 concentration of A. flavus in the diluted rice samples was180 1, 10 or 100 ng/mL. The same procedure was used to181 contaminate the diluted rice samples with 1, 10 or 100 ng/mL182 ofP. chrysogenum and F. oxysporum [24].

183 Development and functionalization of the CNTFETs

184 The SWCNT networks were synthesized on a 500-nm layer185 of silicon dioxide thermally grown on highly doped n-type186 silicon chips (total area 0.5 cm0.5 cm) using chemical187 vapour deposition (CVD). Synthesized SWNTs have been188 characterized using both atomic force microscopy and189 electrical techniques. This process has been described190 elsewhere [27, 28]. The analysis has shown that a dense191 network of SWCNTs with an average height of 1.5 nm is192 commonly obtained after the CVD process. The electrical193 characterization has shown that both metallic and semicon-194 ducting behaviours displayed by the synthesized SWCNTs195 are 30% and 70%, respectively, with RON/OFF~3. To obtain196 the field effect transistor configuration, source and drain197 electrodes were screen-printed with silver ink over the198 synthesized SWCNTs. The gap between both electrodes199 was 0.5 mm, and the size of the electrodes was 200 m200 200 m. The gate electrode was an aluminium layer on201 the back side of Si. The CNTFETs were electrically202 characterized by recording the current vs. the gate203 voltage. To obtain the instrumental variability, we took204 all the electrical characterization measurements three205 times and plotted the mean value and the range value206 of the measurements.207 The functionalization protocol was similar to that208 applied in a previous work where we characterized the209 biofunctionalized layer deposited on the SWCNTs [29].210 Briefly, CNTFETs were incubated for 30 min at 37 C in a211 5 g/mL solution of protein G. This is a small globular cell

212surface protein produced by Streptococcus spp. It is213composed of two or three nearly identical domains of 55214amino acids each. The protein G used in our assays is215genetically truncated, which means that it retains its216affinity for the constant fraction Fc of the IgG but217lacks the Fab-binding sites. Subsequently, the devices218were immersed for 3 h in a solution of Tween 20 and219gelatine (PBSTG). The amount of Tween 20 and gelatine220was optimized to avoid the NSB of the anti-Aspergillus221antibodies and, more importantly, to avoid that the222components of the rice samples could interfere with the223determination of A. flavus. Next, the CNTFETs were224immersed in an 8 g/mL solution of anti-Aspergillus225antibodies for 30 min at 37 C. Finally, the CNTFETs226were again thoroughly rinsed with distilled water and227ready to be used to detect A. flavus. After each228functionalization step, the devices were dried with229nitrogen and electrically characterized. Figure 1 shows230the experimental process used to functionalize the231CNTFET for the detection of A. flavus.

232Optimization of the PBSTG and the dilution factor of rice

233Several CNTFET devices were incubated with protein G.234Each device was then incubated with a different235concentration of Tween 20 and gelatine for 3 h. After236this, the devices were immersed in an 8 g/mL solution237of anti-Aspergillus antibodies for 30 min at 37 C. Finally,238the devices were exposed for 1 h to the diluted rice239samples: first to the 105-fold diluted rice sample and240finally to the 10-fold diluted rice sample.

241Determination of the response time

242A functionalized CNTFET (protected with 1.5% Tween and2432% gelatine) was immersed at 37 C for 1 h in a 103-fold244diluted rice solution contaminated with 1 ng/mL of A.245flavus. Every 15 min, the CNTFET was thoroughly rinsed246with water, dried with nitrogen, electrically characterized247and submerged again in the solution containing 1 ng/mL of248A. flavus for another 15 min.

249Detection of A. flavus

250Another functionalized CNTFET (protected with 1.5%251Tween and 2% gelatine) was exposed to 103-fold diluted252rice samples spiked with increasing concentrations of A.253flavus (i.e. 1, 10 and 100 ng/mL). For each concentration,254the CNTFETs were immersed for 30 min at 37 C, rinsed255thoroughly with distilled water, dried with nitrogen and256electrically characterized. The presence of the moulds in257the recognition layer of the biosensor was confirmed258microscopically with E-SEM.

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259 Selectivity of the CNTFETs

260 Selectivity was checked in the presence of two mycotoxi-261 genic mould P. chrysogenum and F. oxysporum. A262 functionalized CNTFET (protected with 1.5% Tween and263 2% gelatine) was exposed to rice samples contaminated264 with 100 ng/mL of P. chrysogenum, for 30 min at 37 C,265 thoroughly rinsed with distilled water, dried with nitrogen266 and electrically characterized. It was subsequently exposed267 to 100 ng/mL of A. flavus under the same conditions268 mentioned above. The same procedure was followed for F.269 oxysporum. The presence of the A. flavus and the270 interference moulds was also confirmed microscopically271 with E-SEM.

272 Results and discussion

273 Electrical characterization of the functionalized CNTFETs

274 The electrical behaviour of the CNTFETs was monitored275 after each functionalization step in dry conditions and room276 temperature. We measured three times the dependence of277 the source-drain current, I, on the back gate voltage, Vg, in278 the range +5 V to 5 V. The bias voltage, Vsd, was fixed at279 250 mV. Each electrical current plotted corresponds to the280 mean value of three replicates. The change in the electrical281 current as a consequence of the attached proteins has been282 already studied [29].

283 Optimization of the PBSTG and the dilution factor of rice

284 It is essential to prevent the NSB of the antibodies on the285 CNTs and, even more important, to protect the CNTs286 against possible interferences from the rice samples.287 Avoiding the NSB of the anti-Aspergillus antibodies allows288 having all the antibodies with the correct orientation. In this289 way, the sensitivity of the devices is improved because all

290the antibodies are able to provide two antigen binding sites291for A. flavus. Most importantly, preventing the NSB of the292rice compounds onto the CNTs ensures that the electrical293characteristics of the devices will only respond to the294presence ofA. flavus. Therefore, we ensure that the devices295will be selective to A. flavus as long as the antibody does296not have cross reactions with other fungi.297Snchez-Acevedo et al. [30] prevented the NSB of small298molecules by protecting the CNTs with a Tween 20 at2990.05% and gelatine at 0.8% diluted in phosphate buffer300solution (PBSTG). Figure 2 shows the results obtained for a301CNTFET device protected with this solution and after being302exposed to a solution of anti-Aspergillus antibodies. It can303 be seen that in fact the PBSTG effectively protects the304sidewalls of the CNTs against the NSB of the proteins.305However, we also have to protect the CNTs against the306effect of the matrix sample to prevent the rice compounds307from interfering with the detection of A. flavus. Figure 3308shows that increasing the percentage of gelatine to 2% and309Tween to 1.5% enables A. flavus to be detected in 103-fold310diluted rice samples. The change in the electrical current311when the devices are exposed to higher dilution is

Fig. 1 Functionalization process to detectA. flavus with CNTFETs. Protein G enables the antibodies to be appropriately orientated while PBSTGavoids the NSB of biomolecules on the transducer. A. flavus is detected by means of the antigenantibody interaction

Fig. 2 Source-drain current vs. gate voltage of a CNTFET coatedwith PBSTG (solid line) and after being exposed to a solution of anti-

Aspergillus antibodies (solid line with x marks)




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312 attributed to the nature of the sample (i.e. the presence of313 polysaccharides such as starch). As a result, a 103-fold314 dilution of milled rice was chosen as the medium to be315 artificially contaminated with A. flavus, assuring that the316 matrix sample would not affect the electrical behaviour of317 devices protected with 2% of gelatine and 1.5% of Tween318 20.

319 Time response

320 The time response was then optimized by immersing321 functionalized CNTFETs (protected with 2% gelatine and322 1.5% Tween 20) in a 103-fold milled-rice solution containing

3231 ng/mL of A. flavus at 37 C for 1 h. Every 15 min, the324CNTFETs were thoroughly rinsed with water, dried with325nitrogen and electrically characterized. Figure 4 shows that326the highest decrease (i.e. about 12%) in current intensity was327obtained after 30 min of incubation. No further decrease was328obtained. We chose a response time of 30 min for our329devices.

330Detection ofA. flavus mycelia with CNTFETs in milled rice

331A functionalized CNTFET (protected with 2% gelatine and3321.5% Tween 20) was then exposed to 103-fold diluted milled333rice artificially contaminated with 1, 10 and 100 ng/mL ofA.334flavus. For each concentration, the CNTFET was immersed335for 30 min at 37 C, rinsed thoroughly with distilled336water, dried with nitrogen and electrically characterized337 by measuring the IV characteristics three times and338 plotting the mean value and range of the measurement339values.340Figure 5 shows the IVcharacteristics (for a Vsd=0.25 V)341of a functionalized CNTFET before and after exposure to 1,34210 and 100 ng/mL of the mould. It can be seen that the343adsorption of A. flavus decreases the conductance of the344devices (i.e. from a 9% decrease for 1 ng/mL to a 30%345decrease for 100 ng/mL). This may be because the antigen346antibody interaction causes a deformation on the SWCNTs347and thus a scattering effect. The anti-Aspergillus antibody is348able to recognize the antigen galactomannan (GM). It is the349major cell wall component present in Aspergillus species and350is made up of a main chain of mannose with galactose side351groups [31].352E-SEM confirmed that the mould was attached to the353SWCNTs. Figure 6 shows an image of a functionalized

Fig. 5 Gate voltage dependence of the source-drain current of atypical functionalized CNTFET before exposure to A. flavus (solidline), after exposure to the blank solution (1,000-fold diluted ricesample) ( short dashed line), and after the exposure to a 1,000-folddiluted rice sample contaminated with A. flavus at 1 ng/mL (dottedline), 10 ng/mL (long dashed line) and 100 ng/mL (dash and dottedline). The inset shows the behaviour of the source-drain current aftereach functionalization step at Vg=5 V. The error bars correspond tothe range of the electrical current measured for the three replicates

Fig. 4 Source-drain current (at Vg=5 V) obtained for a functional-ized device after being exposed to 1 ng/mL ofA. flavus for 15, 30, 45and 60 min. The error bars correspond to the range of the electricalcurrent measured for the three replicates

Fig. 3 Source-drain current (at Vg=5 V) obtained for threefunctionalized CNTFET coated with different concentrations ofPBSTG. CNTFET1: 0.5% Tween 20 and 0.8% gelatine; CNTFET 2:1% Tween 20 and 1.5% gelatine; CNTFET2: 1.5% Tween 20 and 2%gelatine. Each device was exposed to different dilutions of milled rice(from 10,000-fold to 100-fold). Each electrical current plottedcorresponds to the mean value of three replicates. The error bars

correspond to the range of the electrical current measured for the threereplicates

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354 device after being exposed to A. flavus. We observed that355 regardless of the mycelia filtration process, some spores356 remained attached. A. flavus has hyphae that varies in length357 and that is rough with conidia globose to subglobose varying358 from 3.5 to 4.5 m in diameter. GM antigen is produced at359 an early stage in the metabolic activation of resting conidia,360 but it is increasingly expressed during the swollen conidium361 and hypha stages. Since moulds are primarily found as362 asexual spores or dried mycelia on food, the anti-Aspergillus363 antibody used in our assays could recognize the GM in both364 conidia and hypha structures [25].

365Selectivity of the CNTFETs

366The selectivity of our devices was tested in the presence367of two moulds: P. chrysogenum and F. oxysporum. Under368favourable environmental conditions, these mycotoxigenic369moulds can be found together with A. flavus, causing370diseasein grains during plantgrowth or after harvest in storage.371Chemical analyses of the cell wall of F. oxysporum have372shown that these hyphal walls are composed of (N-acetyl)-373glucosamine, glucose, mannose, galactose, uronic acid and374proteins [32]. Thus, although this mould shares some general375features with A. flavus, it should not have any cross reaction376with the anti-Aspergillus antibody. By contrast, the GM is377present in the cell wall of most Penicillium and Aspergillus378species [33]. Therefore, because these two fungi present379similar antigenic epitopes, cross-reactivity would occur with380the antibody.381A functionalized CNTFET was first immersed in a rice382solution containing 100 ng/mL of P. chrysogenum for38330 min at 37 C, thoroughly rinsed with distilled water,384dried with nitrogen and electrically characterized. The385device was then exposed to 100 ng/mL of A. flavus under386the same conditions as mentioned above. We follow the387same procedure with another functionalized CNTFET for388100 ng/mL of F. oxysporum. Figure 7 shows that the389electrical current changes slightly after the CNTFET was390exposed to F. oxysporum whereas it decreases about 16%391after exposure to A. flavus. The slight changes of the392electrical current after exposing the devices to F. oxysporum393are due to the variability of the electrical current. By394contrast, when the devices are exposed to P. chrysogenum,395the electrical current decreases about 3.5% whereas it396decreases by about 20% after exposure to A. flavus.

Fig. 6 An E-SEM image of a functionalized CNTFET after exposure

to A. flavus. The mould is linked to the SWCNT network throughantigenantibody interactions

Fig. 7 a Gate voltage dependence of the source-drain current of afunctionalized CNTFET before exposure to the mould ( solid line) andafter exposure to 100 ng/mL ofF. oxysporum (solid line with x marks);and 100 ng/mL ofA. flavus (dashed line). b Gate voltage dependenceof the source-drain current of a functionalized CNTFET beforeexposure to the mould (solid line) and after exposure to 100 ng/mL

of P. chrysogenum ( solid line with x marks); and 100 ng/mL of A.flavus (dashed line). Each electrical current plotted corresponds to themean value of three replicates. The inset shows the behaviour ofthe source-drain current at Vg =5 V. The error bars correspond tothe electrical current obtained for the three replicates

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397 The change in the electrical current after exposing the398 devices to P. chrysogenum is probably due to the immuno-399 logical similarity or the identity of the galactomannans in400 both moulds. Therefore, the anti-Aspergillus antibody is able401 to recognize cross-reacting epitopes on other fungal cell402 walls that are causing a slight cross-reactivity with the403 commercially available antibody, as has also been observed404 by De Vos et al. [26]. The electrical current plotted405 corresponds to the mean value of the three measurements.406 The error bars of the inset correspond to the range of the407 current values.408 Selectivity was also checkedwith E-SEM. A functionalized409 CNTFET was exposed to 1, 10 and 100 ng/mL of F.410 oxysporum for 30 min each, thoroughly rinsed with water411 and dried with nitrogen. For each concentration, we scanned412 the area between the electrodes (500 m2) with E-SEM. We413 did not observe either conidia or mycelia when the device414 was exposed to the different mould concentrations. The same415 procedure was followed forP. chrysogenum. In this case, we416 did not observe any conidia or mycelia when the device had417 been exposed to 1 and 10 ng/mL, whereas three conidia were418 observed when it was exposed to 100 ng/mL P. chrysogenum419 (thus showing a slight cross reaction between the antibody420 and P. chrysogenum). With F. oxysporum, the sensor421 displayed high selectivity; however, due to the antigenic422 similarity between P. chrysogenum and A. flavus, there was a423 slight cross-reactivity of the commercially available antibody.424 Therefore, a monoclonal antibody against A. flavus would be425 able to overcome this problem.426 Our CNTFETs are then quite fast. Although the427 construction of the sensor takes about 4 h, the measuring428 time required to detect the mycotoxigenic mould takes only429 30 min whereas conventional media require about 6 days to430 interpret the results [4]. The devices can be built in batch,431 obtaining hundreds of them simultaneously. Moreover,432 CNTFETs are selective and the lowest amount of mould433 detected in distilled water is only 1 ng/mL which is better434 than the limit of detection (LOD) obtained using ELISA435 assays (1 g/mL) [23]. However, when our devices are436 applied to a real sample, the LOD increases to ~10 g/g.437 The devices we have developed in this study have438 improved some performance parameters like selectivity439 and sensitivity if we compare with multisensing systems440 technology. For instance, Paolesse et al. [9] used an441 electronic nose and were able to discriminate between442 non-infected and infected samples with two P. chrysogenum443 and Fusarium verticillioides. Although this sensor can444 detect the early stages of grain spoilage by fungi, the445 detection process is qualitative. Moreover, the sensor446 cannot distinguish between different strains of fungi447 because they produce similar volatile compounds, thus448 reducing the selectivity of the device. It is important to449 emphasize that our devices are disposable; this is mainly

450due to the irreversible deformation of the sidewalls of the451carbon nanotubes as a consequence of the attached452 biomolecules. Moreover, due to the proteic nature of453the molecular receptor (antibodies), they can be denatu-454ralized after their use and also because of the potential455 pathogenicity of the mycotoxigenic mould.

456Conclusions

457We have developed a CNTFET device that uses the458recognition ability of the antibodies and the transduction459capacity of carbon nanotubes to detect A. flavus. Under460adequate conditions (protein concentrations, pH, temperature),461the device could selectively detect at least 10 g/g ofA. flavus462in milled rice. This is probably because the use of protein G463 permits the IgG antibodies to be properly oriented. The464transduction power of the carbon nanotubes makes a special465labelling process unnecessary; as a result, no additional466reagent is required, thereby reducing costs. The sensor467devices only measure the change caused by the antigen468antibody interaction; therefore, the system displays high469selectivity and, since it is well protected, it is not affected470by possible interferences from the raw food. Using suitable471molecular receptors, this type of device would be highly472sensitive and could detect any mycotoxigenic mould in grains473and foods in a short time.474

475Acknowledgments We thank the Spanish Ministry of Education and476Science, MEC, for supporting this study with the project grant477CTQ2007-67570. RAV acknowledges the University of Rovira i478Virgili University for providing economic support. RAV would also479like to thank Veronica Beltran from Arrossaires S.A. del Delta de480l'Ebre for providing the rice samples.

481References

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