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

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AUTHOR'S PROOF!

Metadata of the article that will be visualized in OnlineFirst

1 Article Title Rapid detection of Aspergillus flavus in rice using
biofunctionalized carbon nanotube field effect transistors
2 Article Sub- Title
3 Article Copyright - Springer-Verlag 2010
Year (This will be the copyright line in the final PDF)
4 Journal Name Analytical and Bioanalytical Chemistry

5 Family Name Rius

6 Particle
7 Given Name F. Xavier
8 Corresponding Suffix
9 Author Organization Universitat Rovira i Virgili
10 Division Department of Analytical and Organic Chemistry
11 Address Marcellí. Domingo, s/n, Tarragona 43007, Spain
12 e-mail fxavier.rius@urv.cat
13 Family Name Villamizar
14 Particle
15 Given Name Raquel A.
16 Suffix
17 Organization Universitat Rovira i Virgili
18 Division Department of Analytical and Organic Chemistry
Author
19 Address Marcellí. Domingo, s/n, Tarragona 43007, Spain
20 Organization University of Pamplona
21 Division Department of Microbiology
22 Address Km 1, Vía Bucaramanga, N. de S. Colombia,
Pamplona 31080, Spain
23 e-mail
24 Family Name Maroto
25 Particle
26 Given Name Alicia
27 Suffix
28 Organization Universitat Rovira i Virgili
29 Division Department of Analytical and Organic Chemistry
Author
30 Address Marcellí. Domingo, s/n, Tarragona 43007, Spain
31 Organization École Supérieure de Chimie Organique et Minérale
(ESCOM)
32 Division
33 Address EA 4297 TIMR, 1 allée du réseau Jean-Marie
Buckmaster, Compiègne 60200, France
34 e-mail

file://C:\WMS\Springer\Metadata2PDF\temp\HAB03975.htm 7/6/2010
 Springer Metadata to PDF File Page 2 of 2
AUTHOR'S PROOF!

35 Received 3 February 2010

36 Schedule Revised 8 June 2010
37 Accepted 28 June 2010
38 Abstract In the present study, we have used carbon nanotube field effect transistors
(FET) that have been functionalized with protein G and IgG to detect
Aspergillus flavus in contaminated milled rice. The adsorbed protein G on the
carbon nanotubes walls enables the IgG anti-Aspergillus antibodies to be well
oriented and therefore to display full antigen binding capacity for fungal
antigens. A solution of Tween 20 and gelatine was used as an effective
blocking agent to prevent the non-specific binding of the antibodies and other
moulds and also to protect the transducer against the interferences present in
the rice samples. Our FET devices were able to detect at least 10 µg/g of A.
flavus in only 30 min. To evaluate the selectivity of our biosensors, Fusarium
oxysporum and Penicillium chrysogenum were tested as potential competing
moulds for A. flavus. We have proved that our devices are highly selective tools
for detecting mycotoxigenic moulds at low concentrations in real samples.
39 Keywords Biosensor - Field effect transistors - Carbon nanotubes - Moulds - Aspergillus
separated by ' - ' flavus
40 Foot note
information

file://C:\WMS\Springer\Metadata2PDF\temp\HAB03975.htm 7/6/2010
AUTHOR'S PROOF! JrnlID 216_ArtID 3975_Proof# 1 - 06/07/2010

Anal Bioanal Chem

DOI 10.1007/s00216-010-3975-2
1
3 ORIGINAL PAPER
2

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

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8 Received: 3 February 2010 / Revised: 8 June 2010 / Accepted: 28 June 2010

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9 # Springer-Verlag 2010

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10
11 Abstract In the present study, we have used carbon Introduction 31

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12 nanotube field effect transistors (FET) that have been
13 functionalized with protein G and IgG to detect Aspergillus Aspergillus flavus is a mycotoxigenic and filamentous 32
14 flavus in contaminated milled rice. The adsorbed protein G D mould widely distributed in nature on a variety of food 33
15 on the carbon nanotubes walls enables the IgG anti- and agricultural products. It causes a wide range of 34
16 35
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Aspergillus antibodies to be well oriented and therefore to diseases, ranging from hypersensitive reactions to invasive
17 display full antigen binding capacity for fungal antigens. A infections associated with angioinvasion. A. flavus is after 36
18 solution of Tween 20 and gelatine was used as an effective Aspergillus fumigatus, the second leading cause of invasive 37
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19 blocking agent to prevent the non-specific binding of the and non-invasive aspergillosis [1]. In addition, it is one of 38
20 antibodies and other moulds and also to protect the the most significant fungi in the spoilage of grain during 39
21 transducer against the interferences present in the rice storage and is therefore responsible for the economic 40
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22 samples. Our FET devices were able to detect at least devaluation of the grain through mycotoxin contamination 41
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23 10 μg/g of A. flavus in only 30 min. To evaluate the [2]. Rice is one of the most important staple foods for a 42
24 selectivity of our biosensors, Fusarium oxysporum and large part of the world's human population. It can grow in 43
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25 Penicillium chrysogenum were tested as potential competing different agro-climatic conditions and is vulnerable to 44
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26 moulds for A. flavus. We have proved that our devices are Aspergillus infection in the field as well as in storage. 45
27 highly selective tools for detecting mycotoxigenic moulds at It is difficult to develop a general method for detecting 46
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28 low concentrations in real samples. all mycotoxins because they are metabolites that display 47
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different chemical structures. A good approach, therefore, is to 48

29 Keywords Biosensor . Field effect transistors . Carbon detect the mycotoxigenic moulds in the early stages of growth 49
30 nanotubes . Moulds . Aspergillus flavus before they can produce mycotoxins [3]. Conventional 50
methods for identifying and detecting mycotoxigenic fungi 51
R. A. Villamizar : A. Maroto : F. X. Rius (*) in rice and foods use cultures in different media or 52
Department of Analytical and Organic Chemistry, Universitat immunological methods [4]. Viable plate count method uses 53
Rovira i Virgili,
specific media which allow the moulds to grow over a 54
Marcellí. Domingo, s/n,
43007 Tarragona, Spain surface. A high mould count indicates possible aflatoxin 55
e-mail: fxavier.rius@urv.cat contamination [3]. Other methods currently available analyse 56
the electrical properties of the contaminated food substratum 57
A. Maroto
which are related with the mould grow. Indirect conductance 58
École Supérieure de Chimie Organique et Minérale (ESCOM),
EA 4297 TIMR, 1 allée du réseau Jean-Marie Buckmaster, measurement can be performed and correlated with the 59
60200 Compiègne, France amount of food spoilage yeast [5]. 60
Polymerase chain reaction (PCR)-based methods are 61
R. A. Villamizar
highly specific and have been used to detect aflatoxigenic 62
Department of Microbiology, University of Pamplona,
Km 1, Vía Bucaramanga, N. de S. Colombia, strains of A. flavus [6, 7]. Passone et al. [8] developed a 63
31080 Pamplona, Spain real-time PCR (RT-PCR), directed against the nor-1 gene of 64
AUTHOR'S PROOF!
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R.A. Villamizar et al.

65 the aflatoxin biosynthetic pathway, to monitor and moulds and also to protect the transducer against the 118
66 quantify Aspergillus section Flavi population in peanuts. interferences present in rice samples. The sensor 119
67 Sensitivity tests demonstrated that the test can detect DNA showed high selectivity in the presence of competing 120
68 amounts accounting for a single conidium of Aspergillus moulds. Our devices are label free and able to detect 121
69 parasiticus. It is a rapid, sensitive and specific assay, not 10 μg/g of A. flavus with a time response of 30 min. All 122
70 inhibited by matrix effects. However, it requires the use of these performance parameters are clearly better than those 123
71 a convenient DNA extraction procedure in order to obtain of the methods currently used to detect mycotoxigenic 124
72 reliable results; as a consequence, the method is time fungi. 125
73 consuming.
74 Immunological methods like enzyme-linked immuno-
75 sorbent assay (ELISA) rely on the specific binding of an Materials and methods 126
76 antibody to an antigen. This technique is based on the
77 detection of mycelial antigens or extracellular antigens Proteins and biochemicals 127
78 secreted by moulds with detection range of 1–100 μg/mL

F
79 [3]. However, they require the use of specific labels. Anti-Aspergillus (i.e. a polyclonal rabbit anti-Aspergillus; 128
80 Electronic noses are used to detect volatiles and odours 1 mg/mL) was purchased from Oxford Biotechnology Ltd. 129

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81 produced by the fungi [9–12]. These results can subsequently (Oxford, UK). It was dissolved in PBS to a final 130

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82 be correlated with certain parameters such as mycelia concentration of 8 μg/mL (pH=7.2) and stored at −20 °C 131
83 development on cereal grain or mycotoxin production [9]. until use. Protein G from Streptococcus sp. recombinant, 132

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84 This methodology is able to distinguish between infected expressed in Escherichia coli (1 mg/mL), PBS (pH 7.4), 133
85 and non-infected samples and can sometimes even distinguish Tween 20 and gelatin from cold-water fish skin were 134
86 between non-mycotoxigenic and mycotoxigenic fungi. How- obtained from Sigma-Aldrich. Sabouraud dextrose agar
D 135
87 ever, the methodology is time consuming because the devices (SDA) was provided by oxoid, and brain heart infusion 136
88 137
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have to be previously trained to avoid false-positive or false- (BHI) broth and buffered peptone water were obtained from
89 negative results. Moreover, the information obtained is often Scharlau Chemie Microbiology and prepared according to 138
90 qualitative. their specifications. 139
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91 To overcome this problem, mass and electrochemical

92 sensors have been developed to quickly detect and quantify Apparatus 140
93 fungi [13–15]. Nugaeva et al. [14] used protein-modified
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94 microcantilevers to detect in situ the growth process of A mini-orbital shaker Stuart was used to prepare the fungal 141
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95 Aspergillus niger and Saccharomyces cerevisiae. After 4 h biomass. An environmental scanning electron microscope 142
96 of incubation, the cantilever detected the presence of the (E-SEM), Quanta 600 (FEI, Hillsboro, OR, USA), was used 143
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97 fungi and was able to distinguish between alive and dead to take images of the as-grown networks of SWCNTs and 144
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98 cells. Despite the emerging methods, some performance the functionalization process. Electrical measurements were 145
99 parameters such as sensitivity and selectivity must be taken using a 4157A Agilent semiconductor parameter 146
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100 improved. analyser and a Wentworth Laboratories MP1008 probe 147

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101 Single-walled carbon nanotubes (SWCNTs) are one- station. 148

102 dimensional nanostructures that display remarkable phys-
103 ical and mechanical properties. Moreover, they can be Fungal growth and preparation of fungal biomass 149
104 incorporated into a carbon nanotube field effect transistor
105 (CNTFET) to make a biosensor with improved perfor- Lyophilized strains of A. flavus CECT 2684, Penicillium 150
106 mance parameters [16–21]. In this study, we used a new chrysogenum CECT 2307 and Fusarium oxysporum 151
107 electrochemical biosensor based on carbon nanotube field CECT 2154 were obtained from the Spanish Type Culture 152
108 effect transistors to quickly and selectively detect A. flavus Collection (Valencia, Spain). They were rehydrated with 153
109 in rice samples. In order to improve the sensitivity of our sterile water, subcultured at least three times on SDA and 154
110 devices, we have used protein G; this allows the proper incubated at 30 °C for 7 days to form single colonies [23]. 155
111 orientation of the polyclonal anti-Aspergillus antibodies The spores from the subcultured media were washed from 156
112 and, therefore, optimal antigen binding. Thus, anti- the surface with a PBS solution. Subsequently, flasks 157
113 Aspergillus antibodies are able to provide two specific containing 100 mL of BHI were inoculated with 1 mL of 158
114 binding sites for fungal membrane antigens. A phosphate the spore suspension obtained before and were incubated 159
115 buffer saline (PBS) solution containing Tween 20 and at 30 °C and agitated at 120 rpm for 7 days [24]. The 160
116 gelatine was used as a blocking agent to prevent the non- cultures were harvested and the mycelium was separated 161
117 specific binding (NSB) [22] of the antibodies and other by filtration through Whatman No. 5 filter paper [25]. The 162
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Rapid detection of A. flavus in rice by biofunctionalized CNTFET

Q1
163 mycelium was collected, washed with sterile distilled surface protein produced by Streptococcus spp. It is 212
164 water and dried in the oven at 105 °C until its weight was composed of two or three nearly identical domains of 55 213
165 stable [26]. Then, it was diluted in PBS (w/v) to produce amino acids each. The protein G used in our assays is 214
166 the final samples of A. flavus at 10, 100 and 1,000 ng/mL. genetically truncated, which means that it retains its 215
167 The same procedure was followed for P. chrysogenum and affinity for the constant fraction “Fc” of the IgG but 216
168 F. oxysporum. lacks the Fab-binding sites. Subsequently, the devices 217
were immersed for 3 h in a solution of Tween 20 and 218
169 Rice sample preparation gelatine (PBSTG). The amount of Tween 20 and gelatine 219
was optimized to avoid the NSB of the anti-Aspergillus 220
170 Milled-rice samples were obtained from Arrosaires del antibodies and, more importantly, to avoid that the 221
171 Delta de l´Ebre, Tarragona, Spain. Twenty-five grammes components of the rice samples could interfere with the 222
172 of the sample was dissolved in 225 mL of 0.1% sterile determination of A. flavus. Next, the CNTFETs were 223
173 buffered peptone water and sterilized by autoclaving at immersed in an 8 μg/mL solution of anti-Aspergillus 224
174 121 °C for 15 min. The rice sample was then shaken and antibodies for 30 min at 37 °C. Finally, the CNTFETs 225

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175 diluted in the same solvent for up to 105-fold. After that, were again thoroughly rinsed with distilled water and 226
176 the resulting samples were artificially contaminated by ready to be used to detect A. flavus. After each 227

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177 spiking 1 mL of A. flavus at 10, 100 or 1,000 ng/mL in functionalization step, the devices were dried with 228

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178 9 mL of the diluted rice sample. In this way, the final nitrogen and electrically characterized. Figure 1 shows 229
179 concentration of A. flavus in the diluted rice samples was the experimental process used to functionalize the 230

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180 1, 10 or 100 ng/mL. The same procedure was used to CNTFET for the detection of A. flavus. 231
181 contaminate the diluted rice samples with 1, 10 or 100 ng/mL
182 of P. chrysogenum and F. oxysporum [24]. Optimization of the PBSTG and the dilution factor of rice
D 232

183 233
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Development and functionalization of the CNTFETs Several CNTFET devices were incubated with protein G.
Each device was then incubated with a different 234
184 The SWCNT networks were synthesized on a 500-nm layer concentration of Tween 20 and gelatine for 3 h. After 235
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185 of silicon dioxide thermally grown on highly doped n-type this, the devices were immersed in an 8 μg/mL solution 236
186 silicon chips (total area 0.5 cm×0.5 cm) using chemical of anti-Aspergillus antibodies for 30 min at 37 °C. Finally, 237
187 vapour deposition (CVD). Synthesized SWNTs have been the devices were exposed for 1 h to the diluted rice 238
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188 characterized using both atomic force microscopy and samples: first to the 105-fold diluted rice sample and 239
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189 electrical techniques. This process has been described finally to the 10-fold diluted rice sample. 240
190 elsewhere [27, 28]. The analysis has shown that a dense
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191 network of SWCNTs with an average height of 1.5 nm is Determination of the response time 241
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192 commonly obtained after the CVD process. The electrical

193 characterization has shown that both metallic and semicon- A functionalized CNTFET (protected with 1.5% Tween and 242
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194 ducting behaviours displayed by the synthesized SWCNTs 2% gelatine) was immersed at 37 °C for 1 h in a 103-fold 243
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195 are 30% and 70%, respectively, with RON/OFF ~3. To obtain diluted rice solution contaminated with 1 ng/mL of A. 244
196 the field effect transistor configuration, source and drain flavus. Every 15 min, the CNTFET was thoroughly rinsed 245
197 electrodes were screen-printed with silver ink over the with water, dried with nitrogen, electrically characterized 246
198 synthesized SWCNTs. The gap between both electrodes and submerged again in the solution containing 1 ng/mL of 247
199 was 0.5 mm, and the size of the electrodes was 200 μm× A. flavus for another 15 min. 248
200 200 μm. The gate electrode was an aluminium layer on
201 the back side of Si. The CNTFETs were electrically Detection of A. flavus 249
202 characterized by recording the current vs. the gate
203 voltage. To obtain the instrumental variability, we took Another functionalized CNTFET (protected with 1.5% 250
204 all the electrical characterization measurements three Tween and 2% gelatine) was exposed to 103-fold diluted 251
205 times and plotted the mean value and the range value rice samples spiked with increasing concentrations of A. 252
206 of the measurements. flavus (i.e. 1, 10 and 100 ng/mL). For each concentration, 253
207 The functionalization protocol was similar to that the CNTFETs were immersed for 30 min at 37 °C, rinsed 254
208 applied in a previous work where we characterized the thoroughly with distilled water, dried with nitrogen and 255
209 biofunctionalized layer deposited on the SWCNTs [29]. electrically characterized. The presence of the moulds in 256
210 Briefly, CNTFETs were incubated for 30 min at 37 °C in a the recognition layer of the biosensor was confirmed 257
211 5 μg/mL solution of protein G. This is a small globular cell microscopically with E-SEM. 258
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Q2

Fig. 1 Functionalization process to detect A. flavus with CNTFETs. Protein G enables the antibodies to be appropriately orientated while PBSTG
avoids the NSB of biomolecules on the transducer. A. flavus is detected by means of the antigen–antibody interaction

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259 Selectivity of the CNTFETs the antibodies are able to provide two antigen binding sites 290

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for A. flavus. Most importantly, preventing the NSB of the 291

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260 Selectivity was checked in the presence of two mycotoxi- rice compounds onto the CNTs ensures that the electrical 292
261 genic mould P. chrysogenum and F. oxysporum. A characteristics of the devices will only respond to the 293

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262 functionalized CNTFET (protected with 1.5% Tween and presence of A. flavus. Therefore, we ensure that the devices 294
263 2% gelatine) was exposed to rice samples contaminated will be selective to A. flavus as long as the antibody does 295
264 with 100 ng/mL of P. chrysogenum, for 30 min at 37 °C, not have cross reactions with other fungi. 296
265 thoroughly rinsed with distilled water, dried with nitrogen
D Sánchez-Acevedo et al. [30] prevented the NSB of small 297
266 298
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and electrically characterized. It was subsequently exposed molecules by protecting the CNTs with a Tween 20 at
267 to 100 ng/mL of A. flavus under the same conditions 0.05% and gelatine at 0.8% diluted in phosphate buffer 299
268 mentioned above. The same procedure was followed for F. solution (PBSTG). Figure 2 shows the results obtained for a 300
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269 oxysporum. The presence of the A. flavus and the CNTFET device protected with this solution and after being 301
270 interference moulds was also confirmed microscopically exposed to a solution of anti-Aspergillus antibodies. It can 302
271 with E-SEM. be seen that in fact the PBSTG effectively protects the 303
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sidewalls of the CNTs against the NSB of the proteins. 304

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However, we also have to protect the CNTs against the 305

272 Results and discussion effect of the matrix sample to prevent the rice compounds 306
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from interfering with the detection of A. flavus. Figure 3 307

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273 Electrical characterization of the functionalized CNTFETs shows that increasing the percentage of gelatine to 2% and 308
Tween to 1.5% enables A. flavus to be detected in 103-fold 309
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274 The electrical behaviour of the CNTFETs was monitored diluted rice samples. The change in the electrical current 310
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275 after each functionalization step in dry conditions and room when the devices are exposed to higher dilution is 311
276 temperature. We measured three times the dependence of
277 the source-drain current, I, on the back gate voltage, Vg, in
278 the range +5 V to −5 V. The bias voltage, Vsd, was fixed at
279 250 mV. Each electrical current plotted corresponds to the
280 mean value of three replicates. The change in the electrical
281 current as a consequence of the attached proteins has been
282 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 the

285 CNTs and, even more important, to protect the CNTs
286 against possible interferences from the rice samples.
287 Avoiding the NSB of the anti-Aspergillus antibodies allows
Fig. 2 Source-drain current vs. gate voltage of a CNTFET coated Q3
288 having all the antibodies with the correct orientation. In this with PBSTG (solid line) and after being exposed to a solution of anti-
289 way, the sensitivity of the devices is improved because all Aspergillus antibodies (solid line with x marks)
AUTHOR'S PROOF! JrnlID 216_ArtID 3975_Proof# 1 - 06/07/2010

Rapid detection of A. flavus in rice by biofunctionalized CNTFET

Q1
1 ng/mL of A. flavus at 37 °C for 1 h. Every 15 min, the 323
CNTFETs were thoroughly rinsed with water, dried with 324
nitrogen and electrically characterized. Figure 4 shows that 325
the highest decrease (i.e. about 12%) in current intensity was 326
obtained after 30 min of incubation. No further decrease was 327
obtained. We chose a response time of 30 min for our 328
devices. 329

Detection of A. flavus mycelia with CNTFETs in milled rice 330

A functionalized CNTFET (protected with 2% gelatine and 331

1.5% Tween 20) was then exposed to 103-fold diluted milled 332
rice artificially contaminated with 1, 10 and 100 ng/mL of A. 333
Fig. 3 Source-drain current (at Vg= −5 V) obtained for three flavus. For each concentration, the CNTFET was immersed 334

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functionalized CNTFET coated with different concentrations of for 30 min at 37 °C, rinsed thoroughly with distilled 335
PBSTG. CNTFET1: 0.5% Tween 20 and 0.8% gelatine; CNTFET 2:
water, dried with nitrogen and electrically characterized 336

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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 by measuring the I–V characteristics three times and 337

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(from 10,000-fold to 100-fold). Each electrical current plotted plotting the mean value and range of the measurement 338
corresponds to the mean value of three replicates. The error bars values. 339

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correspond to the range of the electrical current measured for the three
Figure 5 shows the I–V characteristics (for a Vsd=0.25 V) 340
replicates
of a functionalized CNTFET before and after exposure to 1, 341
10 and 100 ng/mL of the mould. It can be seen that the
D 342
312 attributed to the nature of the sample (i.e. the presence of adsorption of A. flavus decreases the conductance of the 343
313 polysaccharides such as starch). As a result, a 103-fold 344
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devices (i.e. from a 9% decrease for 1 ng/mL to a 30%
314 dilution of milled rice was chosen as the medium to be decrease for 100 ng/mL). This may be because the antigen– 345
315 artificially contaminated with A. flavus, assuring that the antibody interaction causes a deformation on the SWCNTs 346
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316 matrix sample would not affect the electrical behaviour of and thus a scattering effect. The anti-Aspergillus antibody is 347
317 devices protected with 2% of gelatine and 1.5% of Tween able to recognize the antigen galactomannan (GM). It is the 348
318 20. major cell wall component present in Aspergillus species and 349
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is made up of a main chain of mannose with galactose side 350

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319 Time response groups [31]. 351

E-SEM confirmed that the mould was attached to the 352
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320 The time response was then optimized by immersing SWCNTs. Figure 6 shows an image of a functionalized 353
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321 functionalized CNTFETs (protected with 2% gelatine and

322 1.5% Tween 20) in a 103-fold milled-rice solution containing
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Fig. 5 Gate voltage dependence of the source-drain current of a

Fig. 4 Source-drain current (at Vg=−5 V) obtained for a functional-
ized device after being exposed to 1 ng/mL of A. flavus for 15, 30, 45
and 60 min. The error bars correspond to the range of the electrical
current measured for the three replicates
typical functionalized CNTFET before exposure to A. flavus (solid Q3
line), after exposure to the blank solution (1,000-fold diluted rice
sample) (short dashed line), and after the exposure to a 1,000-fold
diluted rice sample contaminated with A. flavus at 1 ng/mL (dotted
line), 10 ng/mL (long dashed line) and 100 ng/mL (dash and dotted
line). The inset shows the behaviour of the source-drain current after
each functionalization step at Vg=−5 V. The error bars correspond to
the range of the electrical current measured for the three replicates
AUTHOR'S PROOF!
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Selectivity of the CNTFETs 365

The selectivity of our devices was tested in the presence 366

of two moulds: P. chrysogenum and F. oxysporum. Under 367
favourable environmental conditions, these mycotoxigenic 368
moulds can be found together with A. flavus, causing 369
disease in grains during plant growth or after harvest in storage. 370
Chemical analyses of the cell wall of F. oxysporum have 371
shown that these hyphal walls are composed of (N-acetyl)- 372
glucosamine, glucose, mannose, galactose, uronic acid and 373
proteins [32]. Thus, although this mould shares some general 374
features with A. flavus, it should not have any cross reaction 375
with the anti-Aspergillus antibody. By contrast, the GM is 376
present in the cell wall of most Penicillium and Aspergillus 377

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species [33]. Therefore, because these two fungi present 378
similar antigenic epitopes, cross-reactivity would occur with 379

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the antibody. 380

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A functionalized CNTFET was first immersed in a rice 381
Fig. 6 An E-SEM image of a functionalized CNTFET after exposure solution containing 100 ng/mL of P. chrysogenum for 382

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to A. flavus. The mould is linked to the SWCNT network through 30 min at 37 °C, thoroughly rinsed with distilled water, 383
antigen–antibody interactions
dried with nitrogen and electrically characterized. The 384
device was then exposed to 100 ng/mL of A. flavus under
D 385
354 device after being exposed to A. flavus. We observed that the same conditions as mentioned above. We follow the 386
355 387
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regardless of the mycelia filtration process, some spores same procedure with another functionalized CNTFET for
356 remained attached. A. flavus has hyphae that varies in length 100 ng/mL of F. oxysporum. Figure 7 shows that the 388
357 and that is rough with conidia globose to subglobose varying electrical current changes slightly after the CNTFET was 389
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358 from 3.5 to 4.5 μm in diameter. GM antigen is produced at exposed to F. oxysporum whereas it decreases about 16% 390
359 an early stage in the metabolic activation of resting conidia, after exposure to A. flavus. The slight changes of the 391
360 but it is increasingly expressed during the swollen conidium electrical current after exposing the devices to F. oxysporum 392
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361 and hypha stages. Since moulds are primarily found as are due to the variability of the electrical current. By 393
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362 asexual spores or dried mycelia on food, the anti-Aspergillus contrast, when the devices are exposed to P. chrysogenum, 394
363 antibody used in our assays could recognize the GM in both the electrical current decreases about 3.5% whereas it 395
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364 conidia and hypha structures [25]. decreases by about 20% after exposure to A. flavus. 396
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Fig. 7 a Gate voltage dependence of the source-drain current of a
functionalized CNTFET before exposure to the mould (solid line) and
after exposure to 100 ng/mL of F. oxysporum (solid line with x marks);
and 100 ng/mL of A. flavus (dashed line). b Gate voltage dependence
of the source-drain current of a functionalized CNTFET before
exposure 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 the
Q3
mean value of three replicates. The inset shows the behaviour of
the source-drain current at Vg=−5 V. The error bars correspond to
the electrical current obtained for the three replicates
AUTHOR'S PROOF! JrnlID 216_ArtID 3975_Proof# 1 - 06/07/2010

Rapid detection of A. flavus in rice by biofunctionalized CNTFET

Q1
397 The change in the electrical current after exposing the due to the irreversible deformation of the sidewalls of the 450
398 devices to P. chrysogenum is probably due to the immuno- carbon nanotubes as a consequence of the attached 451
399 logical similarity or the identity of the galactomannans in biomolecules. Moreover, due to the proteic nature of 452
400 both moulds. Therefore, the anti-Aspergillus antibody is able the molecular receptor (antibodies), they can be denatu- 453
401 to recognize cross-reacting epitopes on other fungal cell ralized after their use and also because of the potential 454
402 walls that are causing a slight cross-reactivity with the pathogenicity of the mycotoxigenic mould. 455
403 commercially available antibody, as has also been observed
404 by De Vos et al. [26]. The electrical current plotted
405 corresponds to the mean value of the three measurements. Conclusions 456
406 The error bars of the inset correspond to the range of the
407 current values. We have developed a CNTFET device that uses the 457
408 Selectivity was also checked with E-SEM. A functionalized recognition ability of the antibodies and the transduction 458
409 CNTFET was exposed to 1, 10 and 100 ng/mL of F. capacity of carbon nanotubes to detect A. flavus. Under 459
410 oxysporum for 30 min each, thoroughly rinsed with water adequate conditions (protein concentrations, pH, temperature), 460
the device could selectively detect at least 10 μg/g of A. flavus

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411 and dried with nitrogen. For each concentration, we scanned 461
412 the area between the electrodes (500 μm2) with E-SEM. We in milled rice. This is probably because the use of protein G 462

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413 did not observe either conidia or mycelia when the device permits the IgG antibodies to be properly oriented. The 463

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414 was exposed to the different mould concentrations. The same transduction power of the carbon nanotubes makes a special 464
415 procedure was followed for P. chrysogenum. In this case, we labelling process unnecessary; as a result, no additional 465

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416 did not observe any conidia or mycelia when the device had reagent is required, thereby reducing costs. The sensor 466
417 been exposed to 1 and 10 ng/mL, whereas three conidia were devices only measure the change caused by the antigen– 467
418 observed when it was exposed to 100 ng/mL P. chrysogenum antibody interaction; therefore, the system displays high
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419 (thus showing a slight cross reaction between the antibody selectivity and, since it is well protected, it is not affected 469
420 470
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and P. chrysogenum). With F. oxysporum, the sensor by possible interferences from the raw food. Using suitable
421 displayed high selectivity; however, due to the antigenic molecular receptors, this type of device would be highly 471
422 similarity between P. chrysogenum and A. flavus, there was a sensitive and could detect any mycotoxigenic mould in grains 472
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423 slight cross-reactivity of the commercially available antibody. and foods in a short time. 473
424 Therefore, a monoclonal antibody against A. flavus would be 474
425 able to overcome this problem. Acknowledgments We thank the Spanish Ministry of Education and 475
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Science, MEC, for supporting this study with the project grant 476
426 Our CNTFETs are then quite fast. Although the
CTQ2007-67570. RAV acknowledges the University of Rovira i 477
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427 construction of the sensor takes about 4 h, the measuring Virgili University for providing economic support. RAV would also 478
428 time required to detect the mycotoxigenic mould takes only 479
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like to thank Veronica Beltran from Arrossaires S.A. del Delta de

429 30 min whereas conventional media require about 6 days to l'Ebre for providing the rice samples. 480
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430 interpret the results [4]. The devices can be built in batch,
431 obtaining hundreds of them simultaneously. Moreover,
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432 CNTFETs are selective and the lowest amount of mould References 481
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433 detected in distilled water is only 1 ng/mL which is better

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435 assays (1 μg/mL) [23]. However, when our devices are
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436 applied to a real sample, the LOD increases to ~10 μg/g. (1999) J Agric Food Chem 47:5267–5272 485
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446 cannot distinguish between different strains of fungi Food Microbiol 138:276–281 497
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449 emphasize that our devices are disposable; this is mainly 10. Keshri G, Magan N (2000) J Appl Microbiol 89:825–833 501
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JrnlID 216_ArtID 3975_Proof# 1 - 06/07/2010

R.A. Villamizar et al.

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AUTHOR'S PROOF!
AUTHOR QUERIES

AUTHOR PLEASE ANSWER ALL QUERIES.

Q1. Please check suggested running head.

Q2. Figures 1, 5 and 7 contain pixilated text and lines. Kindly resupply with better images,
preferably in EPS format. Otherwise, kindly advise whether it is okay to proceed with these.
Q3. Kindly check if the descriptions provided for the symbols in the figure captions are appropriate.
Q4. Figure 7, labels were inserted. Kindly check if okay to proceed.

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