electroelectrochemicaldetectionof salmonella using goldnanoparticles.chemicaldetectionof salmonella...

7/16/2019 ElectroElectrochemicaldetectionof Salmonella using goldnanoparticles.chemicaldetectionof Salmonella Using Goldn…

http://slidepdf.com/reader/full/electroelectrochemicaldetectionof-salmonella-using-goldnanoparticleschemicaldetectionof 1/6

Electrochemical detection of Salmonella using gold nanoparticles

Andre S. Afonso a,b,c, Briza Perez-Lopez a,d, Ronaldo C. Faria b, Luiz H.C. Mattoso c, ManuelaHernandez-Herreroe, Artur Xavier Roig-Sagues e, Marisa Maltez-da Costa a, Arben Merkoc- i a,f ,n

a Nanobioelectronics & Biosensors Group, Catalan Institute of Nanotechnology, CIN2 (ICN-CSIC), Universitat Aut onoma de Barcelona, 08193 Bellaterra, Catalonia, Spainb Departamento de Quımica, Universidade Federal de S ~ao Carlos, CP 676, S ~ao Carlos, S ~ao Paulo, CEP 13565-905, Brazilc Embrapa Instrumentac - ~ao Agropecuaria, Laboratorio Nacional de Nanotecnologia para o Agronegocio, CP 741, S ~ao Carlos, S ~ao Paulo, CEP 13560-970, Brazild LEITAT Technological Center, 08225 Terrasa, Spaine Centre Especial de Recerca Planta de Tecnologia dels Aliments (CERPTA), XaRTA, TECNIO, Departament de Ci encia Animal i dels Aliments, Facultat de Veterin aria, Universitat

Aut onoma de Barcelona, 08193 Bellaterra, Barcelona, Spainf ICREA, Barcelona, Spain

a r t i c l e i n f o

Available online 16 July 2012

Keywords:

Gold nanoparticles

Magneto-immunoassay

Salmonella

Electrochemical detection

Label

a b s t r a c t

A disposable immunosensor for Salmonella enterica subsp. enterica serovar Typhimurium LT2 (S) detection

using a magneto-immunoassay and gold nanoparticles (AuNPs) as label for electrochemical detection is

developed. The immunosensor is based on the use of a screen-printed carbon electrode (SPCE) that

incorporates a permanent magnet underneath. Salmonella containing samples (i.e. skimmed milk) have

been tested by using anti-Salmonella magnetic beads (MBs-pSAb) as capture phase and sandwiching

afterwards with AuNPs modified antibodies (sSAb-AuNPs) detected using differential pulse voltammetry

(DPV). A detection limit of 143 cells mL À1 and a linear range from 103 to 106 cells mL À1 of Salmonella was

obtained, with a coefficient of variation of about 2.4%. Recoveries of the sensor by spiking skimmed milk

with different quantities of Salmonella of about 83% and 94% for 1.5Â103 and 1.5Â105 cells mL À1 were

obtained, respectively. This AuNPs detection technology combined with magnetic field application reports

a limit of detection lower than the conventional commercial method carried out for comparison purposes

in skimmed milk samples

&

2012 Elsevier B.V. All rights reserved.

1. Introduction

Foodborne disease has been a serious threat to public health

for many years and still remains a public health problem (WHO,

2011). Salmonella is one of the most frequently occurring patho-

gens in food affecting people’s health (Newell et al., 2010). This

bacteria is transmitted to humans mainly through the consumption

of contaminated food of animal origin such as milk, meat and eggs.

According to World Health Organization (WHO) in the United

States of America (USA), for instance, around 76 million cases of

foodborne diseases, resulting in 325,000 hospitalizations and

5,000 deaths, are estimated to occur yearly (WHO, 2011). In

2011, more than 10 outbreaks comprising hundreds of patients

were reported by Centers for Disease Control and Prevention

(CDC) originated in the ingestion of Salmonella-contaminated

food, leading to medical costs of thousands of dollars (CDC, 2011).

The methods recommended by International agencies of food

health control and International Organization of Standardization

for Salmonella detection in food samples (ICMSF, 2002; ISO, 2002)

are the classical culture methods. These methods can give

qualitative and quantitative information, however, a pre-treat-

ment of the samples is needed; furthermore they are greatly

restricted by the assay time at locations in the food processing or

distribution network, to achieve an earlier detection. Further-

more, to perform them, it is necessary to employ highly skilled

people and more than three days, which exclude their use in field

applications. The development of new methodologies with faster

response time, better sensitivity and selectivity and easy multi-

plexing is still a challenge for food hygiene inspection.

In recent years, new technologies have been developed in

order to improve the time of analysis of the traditional culture

detection. These technologies are mainly based on polymerase

chain reaction (PCR) and immunoassays. Moreover, biosensor

technologies have been used as potential alternatives to circum-

vent the bottlenecks of the standard method because they have

rapid response time and furthermore they are sensitive, robust,

portable and easy to use (Liebana et al., 2009a; Liebana et al.,

2009b; Mata et al., 2010; Salam and Tothill, 2009).

Contents lists available at SciVerse ScienceDirect

journal homepage: www.elsevier.com/locate/bios

Biosensors and Bioelectronics

0956-5663/$- see front matter& 2012 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.bios.2012.06.054

n Corresponding author at: Nanobioelectronics & Biosensors Group, Catalan

Institute of Nanotechnology, CIN2 (ICN-CSIC), Universitat Aut onoma de Barcelona,

08193 Bellaterra, Catalonia, Spain. Tel.: þ34 935868014; fax:þ34 935868020.

E-mail address: [email protected] (A. Merkoc-i).

Biosensors and Bioelectronics 40 (2013) 121–126

http://www.elsevier.com/locate/bios


http://localhost/var/www/apps/conversion/tmp/scratch_10/dx.doi.org/10.1016/j.bios.2012.06.054

mailto:[email protected]















The electrochemical detection methods possess several advan-

tages such as easy operation, low cost, high sensitivity, simple

instrument and suitability for portable devices. Currently, we are

observing a noticeable growth in AuNPs as electrochemical label

for immunoassay (De la Escosura-Muniz et al., 2010). This

electrochemical approach is based on the adsorption of AuNPs

on the surface of the electrotransducer, electrooxidation of the

AuNPs to Au(III), and reverse electroreduction to Au(0), which

generates cathodic peak constituting the analytical signal. TheAuNPs as a label in connection to magnetic particles and screen-

printed carbon electrodes (SPCEs) was also shown to be a very

useful alternative for proteins detection (De la Escosura-Muniz

et al., 2011). However, this technology has not been used for the

screening of pathogenic organisms.

Nanomaterials have received special attention in the develop-

ment of novel biosensing systems (Merkoc- i, 2010). Particularly

nanoparticles have shown to bring interesting advantages for

DNA (Merkoc- i et al., 2005), proteins (De la Escosura-Muniz et al.,

2010) and even cells (Perfezou and Merkoc- i., 2012) analysis. Our

group has already shown the effectiveness of AuNPs for ICP-MS

linked (Merkoc-i et al., 2005a) and electrochemical (Pumera et al.,

2005) DNA assays, electrochemical and optical detections of

human IgG (Ambrosi et al., 2007), CA 15-3 glycoprotein (mainly

used to watch patients with breast cancer) (Ambrosi et al., 2010)

and even of human tumor HMy2 cells (De la Escosura-Muniz

et al., 2009).

Herein, a rapid and sensitive strategy for Salmonella detection,

that takes advantages of AuNPs used as labels and magnetic

particles as preconcentrators, is developed and shown to be

effective enough even for real sample applications. In this approach

the bacteria are captured from the samples (i.e. skimmed milk) and

preconcentrated by immunomagnetic separation, followed by

labeling with AuNPs modified with a polyclonal anti-Salmonella

antibody. Then, the modified MBs are captured by applying a

magnetic field below the SPCE used as transducer for the electro-

chemical detection.

Although other electrochemical biosensing strategies for

Salmonella detection based on nanoparticles (Noguera et al.,2011), carbon nanotubes (Zelada-Guillen et al., 2010) etc. have

already been developed (see Table SI-1 in Supporting Information

Section) the proposed AuNPs electrochemical labeling strategy is

previewed to be of special interest for future in field applications

given the robustness of the electrochemical system in general and

that of nanoparticles particularly.

2. Experimental section

2.1. Materials and apparatus

All voltammetric experiments were performed using an elec-

trochemical analyzer Autolab 20 (Eco-Chemie, The Netherlands)connected to a personal computer using a software package GPS

4.9 (General Purpose Electrochemical System). A thermoshaker

TS1 (Biometra) was used to stir the samples operating at con-

trolled temperature. Transmission Electron Microscope (TEM)

images were taken with Jeol JEM-2011 (Jeol Ltd., Japan). Scanning

electrochemical microscopy (SEM) images were acquired using a

Field Emission-Scanning Electron Microscopy (Merlin, Carl Zeiss).

The electrochemical transducers were homemade screen-

printed carbon electrode (SPCEs), which are constituted by three

electrodes in a single strip: carbon working electrode (WE) with

diameter of 3 mm, Ag/AgCl reference electrode (RE) and carbon

counter electrode (CE). A magnet (3 mm in diameter), inserted

under the WE, was also used to accumulate the complex formed

due to magnetic beads modification with anti-Salmonella first

capturing antibody, Salmonella and AuNPs modified with anti-Salmonella rabbit polyclonal second antibody (MBs-pSAb/S/sSAb-

AuNPs) and used later during the electrochemical measurements.

All glassware used in the synthesis of AuNPs was washed with

aqua regia overnight and the rinsed carefully with milli-Q water.

2.2. Reagents and solutions

Anti-Salmonella magnetic beads modified with the first captur-ing antibody (MBs-pSAb) (Prod. no.1 710.02) was purchased from

Dynal Biotech ASA (Oslo, Norway) and Anti-Salmonella rabbit

polyclonal second antibody (sSAb) (Prod. no. 01.91.99) was from

Biogen scientific (Madrid, Spain). Salmonella enterica subsp. enter-

ica serovar Typhimurium LT2 (CECT 722T) and Escherichia coli

K-12 (CECT 433) strains were purchased from ‘‘Coleccion Espa-

nola de Cultivos Tipo (CECT)’’, Bovine serum albumin, Hydrogen

tetrachloroaurate (III) trihydrate (HAuCl4 Á3H2O, 99.9%), triso-

dium citrate, were purchased from Sigma-Aldrich (St. Louis,

MO). Millipore milli-Q water was obtained from purification

system (18.2 M cm). The buffers were prepared in deionized

water: PBS buffer 10 mM pH 7.4 with 2.7 mM KCl, and 137 mM

NaCl; PBS–Tween buffer (PBS buffer pH 7.4 with tween 20% (m/

v)). Samples for SEM analysis were prepared by using glutaralde-

hyde and hexamethyldisilazane (HMDS) microscopy grade solu-

tions, Sigma-Aldrich (Spain). The electrochemical measurements

were performed in a 0.2 M HCl solution. Finally, all reagents and

other inorganic chemicals were supplied by Sigma-Aldrich or

Fluka, unless otherwise stated.

2.3. Bacterial strains, inocula preparation

Freeze-dried cultures of Salmonella and E. coli were revived in

Tryptone Soy Broth (TSB, Oxoid Ltd., Basingstoke, Hampshire, UK).

Stock cultures of both strains were prepared on Tryptone Soy Agar

(TSA, Oxoid), incubated at 37 1C for 24 h and stored at 4 1C for a

maximum time of 9 weeks. Stock cultures were subcultured into

10 mL of TSB and incubated at 37 1C for 20 h. After incubation, the

broth was spread using a disposable loop on TSA plates andincubated at 37 1C for 20–24 h. Subsequently, cell suspensions

were prepared in 10 mL of PBS–Tween to obtain 9.–9.5 log

cells mL À1. Tubes were placed into a boiling water bath (100 1C)

for 15 min and they were cooled to room temperature prior to

immunological testing. To determine the load of cells before the

heat treatment dilutions were prepared in buffered peptone

water (Oxoid). Then, 1 mL of these dilutions was placed as

duplicate in TSA (Oxoid) and incubated at 37 1C for 24 h.

2.4. Synthesis of gold nanoparticles (AuNPs)

The Turkevich synthesis generates AuNPs of 20 nm (Fig. SI-1).

First a solution of 0.508 mL HAuCl4 (1% m/v) in 49.492 milli-Q

water was heated at 150 1C and stirred. When the solution wasboiling, 5 mL of sodium citrate (40 mmol L À1) were added

rapidly. In the next 10 min of heating and stirring the solution

changed its color from pale yellow to red; it was stirred for

another 15 min at 25 1C (De la Escosura-Muniz et al., 2009a) and

after this step the AuNPs were ready to use. AuNPs were

protected from the light and stored at 4 1C.

2.5. Conjugation of anti-Salmonella rabbit polyclonal second

antibody with AuNPs

First, 100 mL of anti-Salmonella rabbit polyclonal second anti-

body (sSAb) (1 mg mL À1) was added with gentle stirring in 1.5 mL

of colloidal gold suspension with pH adjusted to 9.0 using borate

buffer 50 mM. It was incubated for 20 min at 251C and 650 rpm

A.S. Afonso et al. / Biosensors and Bioelectronics 40 (2013) 121 –126 122



and after that, 100 mL BSA 5% (in milli-Q water) was added and

incubated again for 20 min at 25 1C and 650 rpm. Finally, the

suspension of sSAb-AuNPs was centrifuged for 20 min at

14000 rpm at 4 1C and suspended in 1.5 mL of PBS with 0.3% BSA

according to optimization of immunoassay procedure explained

below.

2.6. The magneto immunoassay

The magneto-immunoassay (see schematic presentation in

Fig. 1A) was performed by mixing 500 mL of solution of different

cells of dead Salmonella (diluted in PBS–Tween in Eppendorf

tubes of 1.5 mL) with 10 mL MBs-pSAb. The mixture was incu-

bated for 30 min at 25 1C with 700 rpm to form the MBs-pSAb/S

magneto-immunoconjugate. After this, the MBs-pSAb/S was sepa-

rated from the supernatant by placing the eppendorf tubes in a

magnetic separator for 3 min and then the supernatant was

discarded. The washing step was performed for 2 min with PBS–

Tween at 25 1C (700 rpm) and MBs-pSAb/S were separated from

supernatant. After two washing steps, the MBs-pSAb/S magneto-

immunoconjugate was resuspended with 140mL of AuNPs

modified with Salmonella antibody (sSAb-AuNPs) and incubated

for 35 min at 25 1C and 700 rpm. Afterwards, the formed MBs-pSAb/S/sSAb-AuNPs magneto-immunosandwich was magneti-

cally separated again, and two times washing step performed as

before. Finally MBs-pSAb/S/sSAb-AuNPs was resuspended in

150 mL PBS and used for further electrochemical analysis.

2.7. SEM sample preparation for MBs-pSAb and MBs-pSAb/S

immunoassay

After the incubation of bacteria with MBs-pSAb, as described

above, the MBs-pSAb/S were kept in PBS suspension and treated

with glutaraldehyde solution followed by sequential dehydration

with ethanol and resuspension in HMDS (hexamethyldisilazane)

solution. This protocol is well suited for fixation of bacteria in

suspension. SEM images were acquired after dropping 4 mL of sample onto a 0.5Â0.5 mm2 SiO2 wafer.

2.8. Electrochemical measurements

The MBs-pSAb/S/sSAb-AuNPs magneto-immunosandwich has

been detected by using SPCEs and electrochemical detection

based on AuNPs label signal (see schematic in Fig. 1B). An aliquot

of 25 mL of MBs-pSAb/S/sSAb-AuNPs magneto-immunosandwich

and 25 mL of 0.2 M HCl was inserted onto SPCE surface while

applying a magnetic field below the SPCE. The electrochemical

detection using DPV technique with parameters previously opti-

mized by Ambrosi et al. (2007): DPV was performed by scanning

from þ1.25 to 0 V (step potential 10 mV, modulation amplitude50 mV, scan rate 33.5 mV sÀ1).

3. Results and discussion

3.1. SEM characterization of MBs-pSAb and MBs-pSAb/S

magnetoconjugates

Biological samples often lack the requirements of structure

stability and electron conductivity necessary for high magnification

SEM images, and it is often necessary to apply metalization

procedures that cover the entire sample with a nano/micro layer

of conductive material that hide the low rugosity of small particlesinteracting with the microorganism’s surface. To avoid the possible

SEM artifacts introduced by the mentioned procedures, we applied

another sample preparation protocol that allows a good fixation

of bacteria and proved to be well suited for SEM analysis. SEM

images in Fig. 2 clearly show the immunologic attachment of the

bacteria to the MBs-pSAb forming MBs-pSAb/S magneto conjugate.

Fig. 2A shows the MBs-pSAb before incubation and Fig. 2B that

corresponds to the incubation of MBs-pSAb with 105 cells mL À1

Salmonella, shows aggregates due to interaction between MBs-

pSAb and bacteria. The difference between them is concordant

with the good recognition obtained during electrochemical detec-

tion of Salmonella, even in the presence of E. coli as interfering

bacteria (images are not shown). It is important to point out that

the micrographs of Fig. 2B correspond to fragments of bacteria due

to the thermal treatment used to kill these.

Fig. 1. Schematic (not in scale) of Salmonella detection. (A) Principle of the assay.

In a first step incubation of Salmonella (S) with magnetic beads (MBs) modified

with primary antibodies specific to the bacteria (pSAb) (MBs-pSAb) occurs. During

this step Salmonella is captured and remains in the MBs-pSAb/S conjugate. During

the second step MBs-pSAb/S conjugate is captured through application of a

permanent magnetic field and washed accordingly. Third step consists in the

incubation of MBs-pSAb/S conjugate with gold nanoparticles (AuNPs) modified

with secondary antibodies (sSAb-AuNPs) and captured again through application

of a permanent magnetic field and washed accordingly. (B) Electrochemical

detection of MBs-pSAb/S/sSAb-AuNPs onto SPCE captured with a magnetic field

(step 5) by using DPV technique. Other experimental conditions as described in

the text.

Fig. 2. SEM images of MBs-pSAb: before (A) and after (B) incubation with 105

cells mL À1

of Salmonella. Other experimental conditions as described in the text.




3.2. Optimizations of the immunoassay parameters

The optimization of labeling parameters of MBs-pSAb/S with

sSAb-AuNPs was performed by evaluating incubation times,

blocking agent and different concentrations of sSAb-AuNPs (0.3;

0.6; 1.45; 3.6 and 7.21 mmol L À1 sSAb-AuNPs) for 105 cells mL À1

Salmonella, collected in PBS–Tween. Fig. 3A and B show the

optimization of incubation time (from 10 and 30 min, respec-

tively) between MBs-pSAb/S conjugate and sSAb-AuNPs (in PBS orPBS–Tween with 0%; 0.3% and 1% of blocking agent (BSA)). The

volume of MBs-pSAb for the immunoassay was recommended by

supplier (10 mL). Although all experiments performed were useful

for Salmonella detection an optimum result in terms of non-

specific adsorption, current value and standard deviation was

obtained by using PBS with 0.3% BSA with an incubation time of

30 min. (see Fig. 3B). In this assay, BSA was very important to

reduce unspecific interaction between MBs-pSAb and sSAb-

AuNPs. Later, the influence of the concentration of sSAb-AuNPs

in PBS with 0.3% of BSA to be used during its second immunor-

eaction with bacteria was also evaluated. As shown in Fig. 3C

sSAb-AuNPs concentration slightly affects the immunoreactions

response, mainly on the reproducibility of method. However, an

optimum and reliable signal was achieved for sSAb-AuNPs

1.45 mmol L À1. Thus, PBS with 0.3% BSA was used as buffer in

all experiments for labeling the MBs-pSAb/S with sSAb-AuNPs

(1.45 mmol L À1 for 30 min).

3.3. Immunosensor response towards Salmonella

Fig. 4 shows the Salmonella detection (from sample collected in

PBS–Tween) obtained due to the signal coming from the sSAb-

AuNPs label. The results obtained for the developed immunosen-

sor for increasing concentrations of target (from 102 to 107

cells mL À1) by using DPV technique show a linear response (from

103 to 106 cells mL À1 with r 2¼0.985). For this assay the current

value corresponding to the LOD was estimated by processing five

negative control samples (0 cells mL À1) that were performed in

two different single inter-day assay, obtaining a mean value

of 0.75 mA (n¼5) that corresponds to 143 cells mL À1 with total

time of analysis of 1:30 h. The precision of the method wasevaluated by testing six different samples with 105 cells mL À1

of Salmonella. The coefficient of variation (CV) obtained was

2.4% (n¼6) indicating good reproducibility under the conditions

describes. A comparison of these results with those reported

previously using other methods, based on nanoparticles, showed

an improvement in general of this approach (see Table SI-1

Supporting information).

3.4. Specificity study for the immunoassay approach

Once the feasibility of detecting of Salmonella using AuNPs was

demonstrated, this assay shows the specificity of the developed

immunoassay by using PBS–Tween and skimmed milk for evaluat-

ing the response toward the Salmonella target in the presence of E.coli as possible interference (see Fig. 5). The current values

obtained for E. Coli assays (in PBS–Tween and skimmed milk)

show similar values (0.85 and 0.98 mA, respectively) as the control

(PBS–Tween and skimmed milked without bacteria) assays (0.66

and 0.92 mA, respectively). Thus, as expected, the electrochemical

signal obtained for E. coli was almost 85% lower than the one

corresponding to Salmonella in both performed assays (PBS–Tween

and skimmed milk). However, for E. Coli assay in PBS–Tween an

Fig. 3. Optimization of the immunoassay approach with sSAb-AuNPs in PBS; PBSþBSA 1%; PBSþBSA 0.3%; PBS–Tween (PBST); PBSTþBSA 1% or PBSTþBSA 0.3%. This

assay was performed with incubation times of 10 min (A) and 30 min (B). Influence of the different concentration of sSAb-AuNPs diluted in PBS with 0.3% BSA onto the

immunoassay response (C). For these assays, 105 cells mL À1 of Salmonella were used. Other experimental conditions as described in the text.

Fig. 4. (A) Typical DPV curves obtained using AuNPs electrochemical detection; (1) immunoassay without Salmonella, (2) 102, (3) 103, (4) 104, (5) 105, (6) 107, (7) 106

cells mL À1 of bacteria. (B) Electrochemical results obtained in the range between 102 and 107 of Salmonella. Dot line represent LOD based of 3 times standard deviation

(n¼5) of control (A-1) plus average of control. The error bars show standard deviation for n¼3. Other experimental conditions as described in the text.




increase of the signal of around 28% was observed. This resultexplained also by the suppliers can be related to a certain degree of

cross reactivity and non-specific binding of the used antibody

(Invitrogen, 2011; KPL, 2011). Nevertheless this level of interfer-

ence does not affect the feasibility of the detection. On the other

hand the result obtained for a mixture of both pathogens is similar

with that obtained for the sample spiked only with 107 cells mL À1

Salmonella.

3.5. Analysis of Salmonella in real samples

The immunosensor was applied to determine the level of

Salmonella in skimmed milk. In order to determine the accuracyof the biosensor technology, skimmed milk purchased in local

commerce area was spiked with Salmonella at different concen-

trations. Recoveries of Salmonella in the range of 83% and 94% (see

Table 1) were calculated. These results demonstrate that the

developed method can be a promising alternative to determineSalmonella in skimmed milk. The obtained detection in real

samples (skimmed milk diluted 10Â in PBS–Tween) is much

lower than the one obtained for Salmonella detection in liquid

samples (i.e. skimmed milk) by using standard commercial

methods, resulting in a value of around 106 cells mL À1 (Fung,

2002). The results obtained show that AuNPs based detection

technology combined with a magnetic field application is capable

of detecting Salmonella at lower concentration than by using

other methods reported in the literature (see Fig. SI-2).

4. Conclusions

A specific and rapid electrochemical based magneto-immuno-

sensor for Salmonella detection in food samples by using AuNPs

has been performed. Salmonella has been captured from the

samples of skimmed milk and preconcentrated by immunomag-

netic separation, followed by labeling with AuNPs modified with a

polyclonal anti-Salmonella antibody. The developed immunosen-

sor is able to detect up to 143 cells mL À1

Salmonella at a rathershorter time (up to 1:30 h). The obtained results are better than

those reported previously not only in the response time but also

due to the fact that AuNPs are easy to be obtained, modified and

detected. The synergy between the immunoassay and magnetic

particles has led to an enhancement of the sensitivity and

removal of interferences from other species. Finally, this techni-

que of detection is suitable for the rapid and sensitive screening-

out of Salmonella in real samples. Furthermore, it could find

several applications in food, medical and environmental fields

where a rapid, cost-efficient and easy to use device for in-field

applications is required.

Acknowledgements

We acknowledge MICINN (Madrid) for the projects PIB2010JP-

00278 and IT2009-0092, and the NATO Science for Peace and

Security Programme’s support under the project SfP 983807 and

to the Conselho Nacional de Desenvolvimento Cientı fico e Tecnolo-

gico (CNPq), Brasil for the scholarship given to Andre Santiago

Afonso, grant number 200826/2011-5 and also Torres Quevedo

scholarship given to Briza Perez-Lopez.

Appendix A. Supporting information

Supplementary data associated with this article can be found

in the online version at http://dx.doi.org/10.1016/j.bios.2012.06.

054.

References

Ambrosi, A., Airo, F., Merkoc-i, A., 2010. Analytical Chemistry 82, 1151–1156.Ambrosi, A., Castaneda, M.T., Killard, A.J., Smyth, M.R., Alegret, S., Merkoc-i, A.,

2007. Analytical Chemistry 79, 5232–5240.CDC, 2011. /http://www.cdc.gov/salmonella/index.htmlS.De la Escosura-Muni z, A., Maltez-da Costa, M., Merkoc-i, A., 2009a. Biosensors and

Bioelectronics 24, 2475–2482.De la Escosura-Muniz, A., Parolo, C., Maran, F., Merkoc-i, A., 2011. Nanoscale 3,

3350–3356.De la Escosura-Muniz, A., Parolo, C., Merkoc-i, A., 2010. Materials Today 13, 24–34.De la Escosura-Muniz, A., Sanchez-Espinel, C., Dıaz-Freitas, B., Gonzalez-Fernan-

dez, A., Maltez-da Costa, M., Merkoc-i, A., 2009. Analytical Chemistry 81,

10268–10274.Fung, D.Y.C., 2002. Comprehensive Reviews in Food Science and Food Safety 1,1–22.

ICMSF, 2002. Microorganisms in Foods 7—Microbial Testing in Food SafetyManagement, 1 ed. Kluwer Academic/Plenum Publishers, New York.

Invitrogen, 2011. Dynabeadss Anti-Salmonella. /www.invitrogen.comS.ISO, 2002. Microbiology of Food and Animal Feeding Stuffs—Horizontal Method

for the Detection of Salmonella spp., 4 ed. International Organisation forStandardization, Geneva, Switzerland.

KPL, 2011. Affinity Purified Antibody to Salmonella Common Structural Antigen(CSA-1). /www.kpl.comS.

Liebana, S., Lermo, A., Campoy, S., Barbe, J., Alegret, S., Pividori, M.I., 2009a.Analytical Chemistry 81, 5812–5820.

Liebana, S., Lermo, A., Campoy, S., Cortes, M.P., Alegret, S., Pividori, M.I., 2009b.Biosensors and Bioelectronics 25, 510–513.

Mata, D., Bejarano, D., Botero, M.L., Lozano, P., Constanti, M., Katakis, I., 2010.Electrochimica Acta 55, 4261–4266.

Merkoc-i, A., 2010. Biosensors and Bioelectronics 26, 1164–1177.Merkoc-i, A., Aldavert, M., Marin, S., Alegret, S., 2005. TrAC, Trends in Analytical

Chemistry 24, 341–349.

Fig. 5. Specificity study for immunoassay performed in both PBS–Tween (PBST)

and diluted skimmed milk, inoculated with E. coli (107 cells mL À1) and a mix of E.

coli and Salmonella (with 107 cells mL À1 of each bacteria). Control samples

correspond to immunoassays without bacteria. Other experimental conditions as

described in the text.

Table 1

Spike and recovery from milk diluted 1/10 in PBS–Tween artificially inoculated

with Salmonella.

Sample Added Found Recovery

(%)

Current

mean

(l A)7SD

Milk 1.5Â103 cells mL À1 1.23Â103 cells mL À1 83.0 1.0170.01

1.5Â105 cells mL À1 1.41Â105 cells mL À1 94.0 1.2570.02


http://localhost/var/www/apps/conversion/tmp/scratch_10/dx.doi.org/doi:10.1016/j.bios.2012.06.054


http://www.cdc.gov/salmonella/index.html



http://www.invitrogen.com/



http://www.kpl.com/

http://www.kpl.com/

http://www.kpl.com/

http://www.kpl.com/







Merkoc-i, A., Aldavert, M., Tarrason, G., Eritja, R., Alegret, S., 2005a. AnalyticalChemistry 77, 6500–6503.

Newell, D.G., Koopmans, M., Verhoef, L., Duizer, E., Aidara-Kane, A., Sprong, H.,Opsteegh, M., Langelaar, M., Threfall, J., Scheutz, F., van der Giessen, J., Kruse,H., 2010. International Journal of Food Microbiology 139, S3–S15.

Noguera, P.S., Posthuma-Trumpie, G.A., van Tuil, M., van der Wal, F.J., de Boer, A.,Moers, A., van Amerongen, A., 2011. Analytical Chemistry 83, 8531–8536.

Perfezou, M., Turner, A., Merkoc-i, A., 2012. Chemical Society Reviews 41,2606–2622.

Pumera, M., Castaneda, M.T., Pividori, M.I., Eritja, R., Merkoc-i, A., Alegret, S., 2005.

Langmuir 21, 9625–9629.Salam, F., Tothill, I.E., 2009. Biosensors and Bioelectronics 24, 2630–2636.WHO, 2011. Food Safety and Foodborne Illness. /http://www.who.int/mediacen

tre/factsheets/fs237/en/S.Zelada-Guillen, G.A., Bhosale, S.V., Riu, J., Rius, F.X., 2010. Analytical Chemistry 82,

9254–9260.


http://www.who.int/mediacentre/factsheets/fs237/en/






electroelectrochemicaldetectionof salmonella using goldnanoparticles.chemicaldetectionof salmonella...

Documents