Sensors and Actuators B 236 (2016) 825–833

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Sensors and Actuators B: Chemical

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

Electrochemical detection of peanuts at trace levels in foods using a

magnetoimmunosensor for the allergenic protein Ara h 2
Víctor Ruiz-Valdepeñas Montiel a,1 , Alessandro Pellicanò b,1 , Susana Campuzano a,∗ ,
Rebeca Magnolia Torrente-Rodríguez a , Ángel Julio Reviejo a , Maria Stella Cosio b ,
José Manuel Pingarrón a,∗
a
Departamento de Química Analítica, Facultad de CC. Químicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain
b
Department of Food, Environmental and Nutritional Sciences (DEFENS), University of Milan, Via Celoria 2, 20133 Milan, Italy

a r t i c l e i n f o a b s t r a c t

Article history: A highly sensitive disposable amperometric magnetoimmunosensor for the rapid determination of Ara
Received 1 December 2015 h 2 protein, one of the major peanut allergens, is reported. The approach uses a sandwich configuration
Received in revised form 23 January 2016 involving selective capture and detector antibodies and carboxylic acid-modified magnetic beads (HOOC-
Accepted 25 January 2016
MBs). Detector antibodies are labeled with HRP-conjugated secondary antibodies and the MBs bearing
Available online 29 January 2016
the immunoconjugates are magnetically captured on surface of a disposable screen-printed carbon elec-
trode (SPCE). The affinity reactions are monitored amperometrically at −0.20 V (vs a Ag pseudo-reference
Keywords:
electrode) in the presence of hydroquinone (HQ) as electron transfer mediator and upon addition of
Ara h 2
Magnetic beads
H2 O2 as the enzyme substrate. The developed magnetoimmunosensor exhibits a wide range of linearity
Screen-printed electrode between 87 and 10,000 pg mL−1 Ara h 2 with a detection limit of 26 pg mL−1 as well as a great selectivity
Amperometric immunosensor against other non-target proteins. The magnetoimmunosensing platform was successfully applied for the
Food extracts detection of Ara h 2 in different food extracts. After an appropriate sample dilution no matrix effects were
observable. The developed methodology was able to detect trace amounts of the peanut allergen (0.0005%
or 5.0 mg kg−1 ) in wheat flour spiked samples. The results correlated properly with those provided by a
commercial ELISA kit.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction disappear when growing up, this allergy is often a lifelong condi-
tion, the most common food-related cause of fatal allergic reactions
The widespread use of peanut products in food industry, the in Western countries [5] and its prevalence is increasing worldwide
severity of the symptoms even for trace quantities in highly sensi- [6,7].
tized people [1], and the persistence in afflicted people have made To date, there is no cure for peanut allergy and no therapeu-
peanut-provoked allergy a major health concern. Although in most tic treatment is available to reduce the severity of this allergy.
people with peanut allergy, symptoms develop after ingesting less Strict avoidance of peanuts and peanut-ingredients remains the
than 1 peanut, which contains about 200 mg of protein [2], in highly mainstay of management. However, full avoidance of peanuts is
sensitized people, subjective symptoms were reported with doses very difficult because peanut seeds are currently widely used as
as low as 100 ␮g, and objective signs were evident with 2 mg [3,4]. source of human food ingredients due to their high quality pro-
Moreover, unlike other food-induced allergies, e.g., allergies trig- tein (22–30%) and oil content (44–56%) [8]. Moreover, the ingestion
gered by milk or egg proteins which mainly affect children and of peanuts often happens accidentally because of product misla-
beling, rework processes which include peanut containing foods
or cross-contamination during processing [9], which represent a
potential risk for peanut allergic individuals. Thus, reliable methods
∗ Corresponding authors. Fax: +34 913944329. for detection and quantification of peanut allergens are necessary
E-mail addresses: victor [email protected] (V. Ruiz-Valdepeñas Montiel), for ensuring the compliance of food labeling and for improving
[email protected] (A. Pellicanò), [email protected]
consumer protection.
(S. Campuzano), [email protected] (R.M. Torrente-Rodríguez),
[email protected] (Á.J. Reviejo), [email protected] (M.S. Cosio), Over 13 allergenic components identified in peanuts, Ara h 2
[email protected] (J.M. Pingarrón). and Ara h 6 are the best predictors of a severe allergic response
1
These authors have contributed equally to this work.

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0925-4005/© 2016 Elsevier B.V. All rights reserved.
826 V. Ruiz-Valdepeñas Montiel et al. / Sensors and Actuators B 236 (2016) 825–833

[10]. Among both, Ara h 2, a 17.5 kDa 2S albumin protein, which A Bunsen AGT-9 Vortex was used for the homogenization of
contributes 5.9–9.3% to the total protein content of a peanut, has the solutions. A Thermomixer MT100 constant temperature incu-
been identified as the most offending peanut allergen [11] and as bator shaker (Universal Labortechnik) and a magnetic separator
an important predictor of clinical reactivity to peanut [6,8]. Dynal MPC-S (product no. 120.20, Dynal Biotech ASA) were also
The detection of peanut allergens in food products is sometimes employed. Capture of the modified-MBs onto the SPCE surface was
a challenging task since they are often only present unintention- controlled by a neodymium magnet (AIMAN GZ) embedded in a
ally and in trace amounts, or can be masked by compounds of homemade casing of Teflon. Centrifuges Cencom (J.P. Selecta S.A.)
the constituting food matrix. Moreover, insufficient knowledge on and MPW-65R were used in the extraction steps.
threshold levels (established by human oral challenge studies) is All the reagents were of the highest available grade. Sodium
available and, therefore, there is general agreement that the achiev- di-hydrogen phosphate (Cat. No.: SO03331000), di-sodium hydro-
able detection limits need to be as low as possible. Indeed, the gen phosphate (Cat. No.: SO03371000), Tris–HCl (Cat. No.:
analytical community and especially standardization bodies are TR04251000), NaCl (Cat. No.: SO02241000) and KCl (Cat. No.:
looking for validated methods that can detect food allergens at PO02001000) were purchased from Scharlab. Tween® 20 (Cat.
<10 mg kg−1 concentrations [12]. Currently, the main analytical No.: 27,434-8), N-(3-dimethylaminopropyl)-N -ethylcarbodiimide
techniques used to detect peanut allergens can be classified into (EDC, Cat. No.: 171440100), N-hydroxysulfosuccinimide (sulfo-
protein-based or DNA-based assays. Protein-based assays detect NHS, Cat. No.: 438650010), ethanolamine (Cat. No.: E9508),
specific peanut allergen (Ara h 1 or Ara h 2), using enzyme-linked hydroquinone (HQ, Cat. No.: H-9003), hydrogen peroxide (H2 O2 ,
immunosorbent assays (ELISAs), or total soluble peanut proteins. 30%, w/v, Cat. No.: H1009) and albumin from chicken egg white
DNA-based techniques detect the presence of allergens by ampli- (OVA, Cat. No.: A-5503) were purchased from Sigma–Aldrich. 2-(N-
fying a specific DNA fragment of a peanut allergen gene through Morpholino)ethanesulfonic acid (MES, Cat. No.: 1080) and bovine
polymerase chain reaction (PCR). However, false positive results serum albumin (BSA Type VH, Cat. No.: 1066) were purchased from
due to cross-reactivity with other nuts [9] as well as large differ- Gerbu and commercial blocker casein solution (a ready-to-use, PBS
ences in quantitative results obtained with available ELISA kits and solution of 1% w/v purified casein) was purchased from Thermo
the high numbers of replicates for samples or an external stan- Scientific (Cat. No.: 37528). Carboxylic acid-modified MBs (HOOC-
dard required by all the described PCR methods have hindered their MBs, 2.7 ␮m Ø, 10 mg mL−1 , Dynabeads® M-270 Carboxylic Acid,
application to processed foods or complex food matrices [13]. Cat. No.: 14305D) were purchased from Dynal Biotech ASA. The cap-
Electrochemical immunosensors have emerged as powerful ture antibody (Mouse monoclonal IgG1, 1C4, clone 1C4 G4 A9, AbC),
alternatives to the above mentioned techniques for the detection the detector antibody (Polyclonal rabbit antiserum raised against
and quantification of allergenic proteins. However their applica- natural purified Ara h 2, AbD), the Ara h 2 standard components
tions in this field are still scarce [14]. To the best of our knowledge, of the Ara h 2 ELISA Kit (Cat. No.: 1C4/AH2), used for comparison
no electrochemical immunosensor has been so far reported for the purposes, and the Ara h 1 standard (Cat. No.: EL-AH1), were pur-
determination of Ara h 2. This paper describes the first electrochem- chased from Indoor Biotechnologies, Inc. Peroxidase-conjugated
ical immunosensor for the selective and sensitive determination of AffiniPure F(ab )2 Fragment Goat anti-Rabbit IgG (F(ab )2 -HRP), Fc
Ara h 2. The proposed design implies the use of functionalized mag- Fragment Specific was purchased from Jackson Laboratories (Cat.
netic beads (MBs) which have demonstrated to constitute powerful No.: 111-036-046).
tools to construct electrochemical immunosensor and minimize All buffer solutions were prepared with water from Milli-
sample matrix effects in the analysis of complex samples such as pore Milli-Q purification system (18.2 M cm). Phosphate-buffered
food extracts [15,16]. In this particular case, the so called mag- saline (PBS) consisting of 0.01 M phosphate buffer solution contain-
netoimmunosensor is based on a sandwich configuration, using a ing 137 mM NaCl and 2.7 mM KCl; 0.01 M sodium phosphate buffer
matched-pair antibody set for a sandwich Ara h 2 immunoassay. solution consisting of PBS with 0.05% Tween® 20 (pH 7.5, PBST);
The capture antibody is immobilized onto carboxylic acid-modified 0.05 M phosphate buffer, pH 6.0; 0.1 M phosphate buffer, pH 8.0;
magnetic beads (HOOC-MBs) whereas the detector antibody was 0.025 M MES buffer, pH 5.0 and 0.1 M Tris–HCl buffer, pH 7.2. Acti-
labeled with a secondary HRP-conjugated anti-rabbit IgG (F(ab )2 - vation of the HOOC-MBs was carried out with an EDC/sulfo–NHS
HRP). The electrochemical detection of the affinity reactions was mixture solution (50 mg mL−1 each in MES buffer, pH 5.0). The
carried out at disposable screen-printed carbon electrodes (SPCEs) blocking step was accomplished with a 1 M ethanolamine solution
using hydroquinone (HQ) as electron transfer mediator and H2 O2 prepared in a 0.1 M phosphate buffer solution of pH 8.0.
as HRP substrate. The applicability of the developed disposable
magnetoimmunosensor was evaluated by determination of the
endogenous Ara h 2 content in food extracts and peanut spiked 2.2. Modification of MBs
samples at trace level.
A 3-␮L aliquot of the HOOC-MBs suspension was transferred
into a 1.5 mL Eppendorf® tube and the MBs were washed twice
with 50 ␮L MES buffer solution during 10 min under continuous
2. Experimental stirring (950 rpm, 25 ◦ C). Between each step the particles were con-
centrated using a magnet and, after 4 min, the supernatant was
2.1. Materials discarded. The carboxylic groups of MBs were activated by incuba-
tion during 35 min in 25 ␮L of the EDC/sulfo–NHS mixture solution.
Amperometric measurements were performed with a CHI812B The activated MBs were washed twice with 50 ␮L of MES buffer
potentiostat (CH Instruments) controlled by software CHI812B; and re-suspended in 25 ␮L of a 50 ␮g mL−1 antiAra h 2 solution in
screen-printed carbon electrodes (SPCEs) (DRP-110, DropSens), MES buffer. The AbC was captured onto the activated beads at 25 ◦ C
consisting of a 4-mm diameter carbon working electrode, a car- under continuous stirring (950 rpm) during 15 min. Subsequently,
bon counter electrode and an Ag pseudo-reference electrode, were the AbC-modified MBs were washed twice with 50 ␮L of MES buffer
employed as transducers and specific cable connector (ref. DRP- solution. Thereafter, the unreacted activated groups on the MBs
CAC also from DropSens, S.L.) acted as interface between the SPCEs were blocked by adding 25 ␮L of the 1 M ethanolamine solution
and the potentiostat. All measurements were carried out at room in 0.1 M phosphate buffer, pH 8.0, and incubating the suspension
temperature. under continuous stirring (950 rpm) for 60 min at 25 ◦ C. After one
 V. Ruiz-Valdepeñas Montiel et al. / Sensors and Actuators B 236 (2016) 825–833 827

washing step with 50 ␮L of 0.1 M Tris–HCl buffer (pH 7.2) and two Table 1
Optimization of the different experimental variables affecting the performance of
more with 50 ␮L of the commercial blocker casein solution.
the amperometric magnetoimmunosensor for Ara h 2 (by comparison of the S/B
The immunoassay was carried out as follows: the AbC-coated ratio obtained in the presence of 5.0 and 0.0 ng mL−1 Ara h 2). See text for other
MBs were re-suspended in 25 ␮L of a mixture solution prepared in used variables.
commercial blocker casein solution containing the target Ara h 2
Variable Tested range Selected value
(or the sample under study) and the AbD (1/1000) and incubating
during 45 min (950 rpm, 25 ◦ C); after two washing steps with 50 ␮L [AbC], ␮g mL−1 2.5–100 50
tAbC , min 15–90 15
of PBST buffer solution (pH 7.5), the modified MBs were incubated
[AbD], dilution 1/250–1/5000 1/1000
with F(ab )2 -HRP (1/10,000) solution in PBST (pH 7.5) during 30 min [F(ab )2 -HRP], dilution 1/250–1/50,000 1/10,000
(950 rpm, 25 ◦ C). Two additional washings with 50 ␮L of PBST (pH tAg + AbD , min 15–90 45
7.5) were carried out. tF(ab ) 2-HRP , min 15–90 30
Finally, the modified-MBs were re-suspended in 45 ␮L of 0.05 M
sodium phosphate buffer solution (pH 6.0) to perform the amper-
ometric detection. Regarding the analysis of spiked samples, peanut-free wheat
The different procedures tested in the optimization of the num- flour (verified using a commercial Ara h 2 ELISA spectrophotometric
ber of steps involved in the immunoassay procedure were: (1) one kit) was spiked with different amounts of peanut flour that con-
single step involving both Ara h 2 capture, sandwiching with AbD sisted of 100% raw peanut (unknown variety) from a commercial
and labeling with F(ab )2 -HRP by 30 min incubation of the AbC-MBs retailer (Frinuts). Accordingly, a series of mixtures containing 0.05,
in a mixture solution containing Ara h 2, AbD and F(ab )2 -HRP; (2A) 0.025, 0.01, 0.0075, 0.005, 0.001, 0.0005 and 0.0001% w/w of peanut
two steps involving 30 min incubation with the Ara h 2 solution, fol- were prepared.
lowed by another 30 min incubation step in the mixture solution The following protocol was used for the extraction of pro-
containing AbD and F(ab )2 -HRP; (2B) two steps involving 30 min teins present in peanuts in all the food samples analyzed: 0.5 g
incubation in a mixture solution containing Ara h 2 and AbD, fol- of accurately weighted ground sample (previously blended) were
lowed by 30 min incubation in the F(ab )2 -HRP solution; (3) three introduced in plastic tubes and incubated in 5.0 mL of Tris–HCl (pH
steps implying sequential incubations of 30 min in Ara h 2, AbD and 8.2) overnight at 60 ◦ C under continuous stirring (950 rpm). Regard-
F(ab )2 -HRP solutions, respectively. ing chocolate samples, they were frozen at −20 ◦ C before blending,
The storage stability of the AbC-MBs was checked by keeping and 0.5 g of skimmed milk powder (Central Lechera Asturiana® )
them at 4 ◦ C in microcentrifuge tubes containing 50 ␮L of filtered were added during the extraction in order to avoid masking of the
PBS. Two replicates of the stored conjugates were incubated each target protein by tannins [12]. Subsequently the aqueous phase
working day in solutions containing 0 or 2.5 ng mL−1 Ara h 2 fol- was isolated by centrifugation involving a first step at 3,600 rpm
lowing the optimized protocol. A control chart was constructed by during 10 min and a second step at 10,000 rpm during 3 min (4 ◦ C)
setting as the central value the average current value calculated for a 1-mL aliquot of the first supernatant [5,16,18]. The resulting
from 10 measurements made the first day of the study for both Ara supernatant appropriately diluted was used to perform the Ara h 2
h 2 concentration levels, while the upper and lower limits of control determination with the magnetoimmunosensor.
were set at ±3 × SD of these initial values. In order to make comparison, the same food extracts were also
analyzed by applying an ELISA method involving the use of the same
immunoreagents.
2.3. Amperometric measurements

To perform the amperometric measurements the SPCE was 3. Results and discussion
positioned on a homemade casing of Teflon with neodymium
magnet encapsulated, and the 45 ␮L of the modified MBs suspen- The fundamentals of the magnetoimmunosensor configuration
sion were magnetically captured on the SPCE surface according as well as of the electrochemical transduction used in this work are
to the procedure described earlier [17]. Then, the magnet hold- displayed in Fig. 1. As can be observed, all the immunoreactions
ing block was immersed into an electrochemical cell containing involved in the assay occurred on the MBs surface. After magnetic
10 mL of 0.05 M phosphate buffer of pH 6.0 and 1.0 mM HQ (pre- capturing of the MBs bearing the sandwich-type immunoconju-
pared just before performing the electrochemical measurement). gates on the working electrode surface, amperometric detection
Amperometric measurements in stirred solutions were made by of the HRP enzymatically catalyzed reduction current generated in
applying a detection potential of −0.20 V vs Ag pseudo-reference the presence of the HQ/H2 O2 redox system was carried out.
electrode upon addition of 50 ␮L of a 0.1 M H2 O2 solution until
the steady-state current was reached (approx. 100 s). The ampero-
3.1. Optimization of experimental variables
metric signals given through the manuscript corresponded to the
difference between the steady-state and the background currents.
No significant non-specific binding of Ara h 2, AbD and F(ab )2 -
HRP was observed when no AbC was immobilized on HOOC-MBs
2.4. Analysis of real samples and a commercial blocker casein solution was employed. This
blocking solution has proven to be highly effective for minimiza-
The Ara h 2 amperometric magnetoimmunosensor was applied tion of non-specific adsorptions [16]. Therefore, the sandwich
to the analysis of different food samples containing unknown configuration approach was feasible for the target protein determi-
amounts of endogenous Ara h 2 and also samples free of peanuts nation. Subsequently, the optimization of experimental variables
(wheat flour) spiked at trace levels. involved in the magnetoimmunosensor preparation and function-
Different types of foodstuffs, purchased in local supermarkets, ing was accomplished. The adopted criterion of selection for each
were analyzed: wheat flour, hazelnuts; peanuts (peanut flour, raw, variable was the magnitude of the ratio between the amperomet-
fried and chocolate-coated); chocolate bars with roasted peanuts, ric responses measured at −0.20 V in the presence (signal, S) of
nougat, caramel and milk chocolate; multicereals bars with up to 5.0 ng mL−1 Ara h 2 and in the absence of target protein (blank, B)
59% (w/w) of whole roasted peanuts; chocolate chip cookies with (S/B ratio). All the tested variables, the ranges into which they were
caramelized almonds; peanut creams and peanut oil. checked and the selected values for further work are summarized
828 V. Ruiz-Valdepeñas Montiel et al. / Sensors and Actuators B 236 (2016) 825–833

Fig. 1. Schematic display of the Ara h 2 magnetoimmunosensor fundamentals.

Fig. 2. Effect of the number of incubation steps used to perform the sandwich immunoassay for Ara h 2 determination on the amperometric responses measured for 0 (white
bars) and 5 ng mL−1 (grey bars) Ara h 2. Error bars estimated as triple of the standard deviation (n = 3). See additional details in Section 2.2.

in Table 1. The detection potential used and the volume of MBs used 3.2. Analytical characteristics
per assay were optimized in previous works [19,20].
In addition to the experimental variables collected in Table 1 A calibration plot was constructed with Ara h 2 standards under
and with the aim of simplifying as much as possible the whole the selected experimental conditions. As it can be seen in Fig. 3, a
protocol and reducing the assay time, the effect of the number of linear relationship (r = 0.999) between the measured current and
involved working steps in the immunoassay procedure following the Ara h 2 concentration was found over the 87–10,000 pg mL−1
the protocol described in Section 2.2 was evaluated. The obtained range, with a slope and intercept values of (198 ± 2) nA mL ng−1 and
results indicating clearly as the protocol involving an initial incu- (87 ± 8) nA, respectively. The LOD and the determination limit (LQ),
bation step with the mixture solution of the target protein and the 26 and 87 pg mL−1 , respectively, were calculated according to the
AbD, and a latter incubation with F(ab )2 -HRP (bars 2B in Fig. 2) 3s m−1 and 10s m−1 criteria, respectively, where m is the slope of
provided the largest S/B current ratio. This effect was most likely the calibration plot and s was estimated as the standard deviation
due to the lower target recognition efficiency as a consequence of ten amperometric signals obtained without target Ara h 2.
of increased steric hindrance when all the immunoreagents were Amperometric measurements for 2.5 ng mL−1 Ara h 2 carried out
mixed in homogeneous solution (bars 1) or the AbD was incubated with 10 different magnetoimmunosensors prepared using the opti-
together with the F(ab )2 -HRP (bars 2A). It is worth to mention also mized protocol yielded a relative standard deviation (RSD) value of
that the S/B ratio was significantly larger when the target protein 3.3%, thus indicating a great reproducibility of the whole magne-
was incubated with the AbD in the same step (bars 2B) than in the toimmunosensor fabrication and the signal transduction protocols.
case of applying separate steps (bars 3), probably due to a more The results obtained in the storage stability study of the AbC-
efficient binding with AbD when Ara h 2 is free in solution. Accord- MBs (not shown, see additional details in Section 2.2) showed
ingly, the protocol 2B was employed for the implementation of the that the magnetoimmunosensors response remained within the
magnetoimmunosensor. control limits for 50 days. This great storage stability allows the
preparation of AbC-MBs conjugates sets and the possibility of their
 V. Ruiz-Valdepeñas Montiel et al. / Sensors and Actuators B 236 (2016) 825–833 829

Fig. 3. Calibration plot constructed for Ara h 2 standards with the developed magnetoimmunosensor. Error bars were estimated as triple of the standard deviation (n = 3).

Fig. 4. Current values were measured for 0 (white bars) and 2.5 (grey bars) ng mL−1 Ara h 2 in the absence or in the presence of 250 ng mL−1 Ara h 1, 50 mg mL−1 BSA
and 130 mg mL−1 OVA. Supporting electrolyte, 0.05 M sodium phosphate solution, pH 6.0; Eapp = −0.20 V vs Ag pseudo-reference electrode. Other conditions as described in
Table 1 (selected values column). Error bars estimated as triple of the standard deviation (n = 3).

storage under the above mentioned conditions until the magne- observed with the cupin Ara h 1 despite this protein was tested at a
toimmunosensor needs to be prepared. 100 times larger concentration and shows short similar structural
motifs with Ara h 2 [8].

3.3. Selectivity of the magnetoimmunosensor

3.4. Determination of Ara h 2 in food samples
The selectivity of the magnetoimmunosensor was evaluated
both toward Ara h 1, the other major allergen in peanuts, and The real usefulness of the developed methodology was eval-
non-target proteins such as BSA and OVA which can coexist with uated by determining the target allergen protein in different food
the target protein in food extracts. These tests were performed extracts containing variable unknown content of endogenous Ara h
by comparing the current values measured with the magnetoim- 2 as well as in spiked target protein-free samples. The used protocol
munosensor for 0 and 2.5 ng mL−1 Ara h 2 in the absence and is illustrated in Fig. 5.
in the presence of these potential interfering compounds. Fig. 4 No statistically significant differences were observed between
clearly shows as no significant interference was apparent for Ara h the slope value of the calibration plot constructed with Ara h 2
2 determination as a result of the presence of the three tested non- standards and the slope values of the calibration graphs recorded
target proteins even at the large concentrations assayed for BSA and from all the prepared extracts once they were adequately diluted
OVA. It is worth to highlight that no noticeable cross-reactivity was with blocker casein solution and spiked with growing amounts
830 V. Ruiz-Valdepeñas Montiel et al. / Sensors and Actuators B 236 (2016) 825–833

Fig. 5. Schematic display of the protocol used to obtain the food extracts (exemplified for peanuts case) and amperometric traces recorded with the magnetoimmunosensor
for undiluted wheat flour and 1/500,000 diluted-peanut flour extracts prepared following the protocol described in Section 2.4 (a). Comparison of the results obtained with
the magnetoimmunosensor and the ELISA (b). Error bars estimated as triple of the standard deviation (n = 3).

of a standard Ara h 2 solution up to 2.5 ng mL−1 . Accordingly, we Ara h 2 concentrations measured with the ELISA kit and those
concluded that no significant matrix effects were apparent once obtained with the amperometric magnetoimmunosensor (Fig. 5b)
the sample dilution factors required to fit the target analyte con- with the confidence intervals (at a significance level of ˛ = 0.05)
centration into the linear range of the calibration graph shown in for the slope and intercept including the unit and the zero val-
Fig. 3, were applied, confirming also the effectiveness in the use ues, respectively. However, the use of the magnetoimmunosensor
of MBs and blocker casein solution to minimize the non-specific allowed the analysis to be made in half-time compared with
adsorption of components from complex samples. Therefore, Ara h the ELISA method (2 vs 4 h once the AbC-MBs and AbC-plate
2 quantification could be accomplished by simple interpolation of were prepared, respectively), simplified largely the whole analyt-
the measured current from the diluted samples into the calibration ical procedure required smaller sample and bioreagents volumes
plot constructed with Ara h 2 standard solutions. (25 vs 100 ␮L), with the consequent saving in cost per analy-
The results obtained for all the assayed food extracts together sis, and can be easily automated and implemented with portable
with the dilution factor applied to each analyzed extract are pre- and cost-effective instrumentation. These characteristics make
sented in Table 2. In addition, the obtained results were compared the magnetoimmunosensor an attractive and user-friendly tool
with those provided by a commercial ELISA kit using the same to monitor routinely the food quality and to perform decentral-
immunoreagents. An excellent correlation was found between the ized analysis in comparison with the ELISA method which is the
most commonly used by the food industry and official food control
 V. Ruiz-Valdepeñas Montiel et al. / Sensors and Actuators B 236 (2016) 825–833 831

Fig. 6. Amperometric responses measured with Ara h 1 and Ara h 2 magnetoimmunosensors from extracts for unspiked wheat flour and spiked with increasing amounts of
peanut flour (final concentrations: 0.0001, 0.0005, 0.001, 0.005, 0.0075, 0.01, 0.025 and 0.05% (w/w)) (a). Amperometric traces recorded with the Ara h 1 (left) and Ara h 2
(right) magnetoimmunosensor from extracts of unspiked wheat flour and spiked with 0.05% (w/w) (left) or 0.0005% (w/w) peanut flour (right). Error bars were estimated as
triple of the standard deviation (n = 3).

Table 2
Determination of allergen Ara h 2 concentration (in mg g−1 ) in different food extracts using the developed amperometric magnetoimmunosensor and comparison with the
results provided by a commercial ELISA spectrophotometric kit.

Extract Dilution factor ELISA Magnetoimmunosensor

Fried peanuts 1/100,000 (4.4 ± 0.3) (4.3 ± 0.3)

RSDn = 3 = 6.4% RSDn = 3 = 2.7%

Raw peanuts 1/100,000 (4.1 ± 0.4) (4.3 ± 0.4)

RSDn = 3 = 4.3% RSDn = 3 = 3.6%

Chocolate-covered peanuts 1/1000 (0.034 ± 0.006) (0.033 ± 0.005)

RSDn = 3 = 6.5% RSDn = 3 = 6.5%

Chocolate bars with roasted peanuts 1/10,000 (0.38 ± 0.02) (0.31 ± 0.04)
RSDn = 3 = 2.4% RSDn = 3 = 5.5%

Multicereals bars with roasted peanuts 1/100,000 (3.4 ± 0.5) (3.5 ± 0.6)
RSDn = 3 = 6.1% RSDn = 3 = 6.5%

Peanut cream 1 1/100,000 (3.6 ± 0.8) (3.6 ± 0.3)

RSDn = 3 = 8.8% RSDn = 3 = 3.4%

Peanut cream 2 1/100,000 (1.4 ± 0.2) (1.3 ± 0.3)

RSDn = 3 = 6.3% RSDn = 3 = 8.6%

Peanut oil 1/1000 (0.0023 ± 0.0004) (0.0022 ± 0.0004)

RSDn = 3 = 7.0% RSDn = 3 = 6.5%

Peanut flour 1/500,000 (11 ± 1) (10.9 ± 0.7)

RSDn = 3 = 4.0% RSDn = 3 = 2.5%

Wheat flour – ND ND
Raw halzenuts – ND ND

ND: non detectable.

832 V. Ruiz-Valdepeñas Montiel et al. / Sensors and Actuators B 236 (2016) 825–833

agencies to assess the presence of allergens like peanut in food Acknowledgments

products [9].
Regarding the analysis of wheat flour containing no detectable The financial support of the Spanish Ministerio de Economía
content of the target allergen (see Table 2), it was spiked with dif- y Competitividad Research Projects, CTQ2015-64402-C2-1-R, and
ferent increasing amounts of peanut flour and the corresponding the NANOAVANSENS Program from the Comunidad de Madrid
extracts were prepared and analyzed as described in Section 2.4. (S2013/MT-3029) are gratefully acknowledged. Financial support
The results are summarized in Fig. 6, where the measurements by COFIN 2010–2011 (MIUR, 2010 AXENJ8 002) is also acknowl-
made with an Ara h 1 immunosensor previously developed in our edged. R.M. Torrente-Rodríguez acknowledges a CPFD predoctoral
group are also displayed [16]. These results demonstrated that the contract from the Spanish Ministerio de Economía y Competitivi-
Ara h 2 magnetoimmunosensor was able to detect clearly 0.0005% dad.
(5.0 mg kg−1 ) peanut, improving in a factor of 100–200 the low-
est achievable trace peanuts concentration reported previously in
the literature based on Ara h 1 determination, 0.05% [16] and 0.1% References
[14,21]. This highly improved sensitivity for peanut detection can
be attributable to the use of different immunoreagents and label- [1] S. Al-Muhsen, A.E. Clarke, R.S. Kagan, Peanut allergy: an overview, CMAJ 168
(2003) 1279–1285.
ing protocol (use of F(ab )2 -HRP instead of Strep-HRP) and to a [2] M. Goldman, Peanut Allergy: How Much Peanut is Too Much? Baltimore
more efficient extraction of the smaller target protein (17.5 vs (MD): Asthma and Allergy Foundation of America, Maryland—Greater
65 kDa for Ara h 1) from the food samples. This enhanced sensitiv- Washington, DC (Chapter Newsletter; April–May 1998).
[3] J.B. Hourihane, S.A. Kilburn, J.A. Nordlee, S.L. Hefle, S.L. Taylor, J.O. Warner, An
ity represent a major comparative advantage taking into account
evaluation of the sensitivity of subjects with peanut allergy to very low doses
the serious public health problem that the degree of contamina- of peanut protein: a randomized, double-blind, placebo-controlled food
tion of commercial food samples with peanuts, whether fraudulent challenge study, J. Allergy Clin. Immunol. 100 (1997) 596–600.
[4] S.L. Taylor, S.L. Hefle, C. Bindslev-Jensen, S.A. Bock, A.W. Burks Jr., L.L. Christie,
or accidental, can cause in sensitive individuals. Moreover, it is
D.J. Hill, A. Host, J.O’B. Hourihane, G. Lack, D.D. Metcalfe, D.A.
important to emphasize that although a level of 10 mg kg−1 is con- Moneret-Vautrin, P.A. Vadas, F. Rance, D.J. Skrypec, T.A. Trautman, I.
sidered relevant for the detection of potentially hazardous residues Malmheden Yman, R.S. Zeiger, Factors affecting the determination of
of undeclared allergens in foods, the achievement of lower detec- threshold doses for allergenic foods: how much is too much? J. Allergy Clin.
Immunol. 109 (2002) 24–30.
tion limits, as it is the case of this work, may be highly helpful since [5] P. Jollet, F. Delport, K.P.F. Janssen, D.T. Tran, J. Wouters, T. Verbiest, J.
minimal amounts of the target allergen can be critical [9]. In fact, Lammertyn, Fast and accurate peanut allergen detection with nanobead
in most people suffering peanut allergy, symptoms are developed enhanced optical fiber SPR biosensor, Talanta 83 (2011) 1436–1441.
[6] N. Nicolaou, M. Poorafshar, C. Murray, A. Simpson, H. Winell, G. Kerry, A.
after ingesting less than 1 peanut and, in highly sensitized people, Härlin, A. Woodcock, S. Ahlstedt, A. Custovic, Allergy or tolerance in children
subjective symptoms have been reported even with doses as low sensitized to peanut: prevalence and differentiation using
as 100 ␮g [3,4]. component-resolved diagnostics, J. Allergy Clin. Immunol. 125 (2010)
191–197.
Moreover, it is important to mention that the detection level [7] Y. Zhou, J.-s. Wang, X.-j. Yang, D.-h. Lin, Y.-f. Gao, Y.-j. Su, S. Yang, Y.-j. Zhang,
for peanuts achieved with the magnetoimmunosensor, about J.-j. Zheng, Peanut allergy, allergen composition, and methods of reducing
5.0 mg kg−1 , incurred in wheat flour is in the range of some allergenicity: a review, Int. J. Food Sci. (2013) 1–8, http://dx.doi.org/10.1155/
2013/909140, Article ID 909140.
of the most promising PCR-based approaches reported for sen-
[8] M. Bublin, H. Breiteneder, Cross-reactivity of peanut allergens, Curr. Allergy
sitive peanut determination, which are in the 2–10 mg kg−1 Asthma Rep. 14 (2014) 426, http://dx.doi.org/10.1007/s11882-014-0426-8.
range [9,22–24]. Most interestingly, the LOD provided by the [9] I.M. López-Calleja, S. de la Cruz, N. Pegels, I. González, T. García, R. Martín,
Development of a real time PCR assay for detection of allergenic trace
immunosensor is achieved without performing any amplification
amounts of peanut (Arachis hypogaea) in processed foods, Food Control 30
step. (2013) 480–490.
[10] A.K. Kukkonen, A.S. Pelkonen, S. Mäkinen-Kiljunen, H. Voutilainen, M.J.
Mäkelä, Ara h 2 and Ara 6 are the best predictors of severe peanut allergy: a
double-blind placebo-controlled study, Allergy 70 (2015) 1239–1245.
[11] G.W. Palmer, D.A. Dibbern, A.W. Burks, G.A. Bannon, S.A. Bock, H.S. Porterfield,
R.A. McDermott, S.C. Dreskin, Comparative potency of Ara h 1 and Ara h 2 in
4. Conclusions immunochemical and functional assays of allergenicity, Clin. Immunol. 115
(2015) 302–312.
[12] R.E. Poms, M.E. Agazzi, A. Bau, M. Brohee, C. Capelletti, J.V. Nørgaard, E.
A disposable amperometric magnetoimmunosensor for the Anklam, Inter-laboratory validation study of five commercial ELISA test kits
high sensitive determination of the allergenic protein Ara h 2 is for the determination of peanut proteins in biscuits and dark chocolate, Food
described for the first time in this work. This magnetoimmunosen- Addit. Contam. 22 (2005) 104–112.
[13] T. Holzhauser, K. Kleiner, A. Janise, M. Röder, Matrix-normalised
sor allows a LOD of 26 pg mL−1 to be obtained for the target protein quantification of species by threshold-calibrated competitive real-time PCR:
and has clearly demonstrated successful applicability for the accu- allergenic peanut in food as one example, Food Chem. 163 (2014) 68–76.
rate determination of the endogenous content of Ara h 2 in different [14] R.C. Alves, F.B. Pimentel, H.P.A. Nouws, R.C.B. Marques, M.B. González-García,
M.B.P.P. Oliveira, C. Delerue-Matos, Detection of Ara h 1 (a major peanut
food extracts. The achieved results benchmarked against a com- allergen) in food using an electrochemical gold nanoparticle-coated
mercially available ELISA kit and allowed the detection of peanut screen-printed immunosensor, Biosens. Bioelectron. 64 (2015) 19–24.
trace contents (5 mg kg−1 ) in spiked wheat flour samples. The great [15] E. Zacco, M.I. Pividori, S. Alegret, R. Galve, M.P. Marco, Electrochemical
magnetoimmunosensing strategy for the detection of pesticides residues,
analytical performance achieved also in terms of high selectivity, Anal. Chem. 78 (2006) 1780–1788.
accuracy, reliability and reproducibility in complex food matri- [16] V. Ruiz-Valdepeñas Montiel, S. Campuzano, A. Pellicanò, R.M.
ces, together with the flexibility and simplicity of the disposable Torrente-Rodríguez, A.J. Reviejo, M.S. Cosio, J.M. Pingarrón, Sensitive and
selective magnetoimmunosensing platform for determination of the food
magnetoimmunosensor, mean important advantages for an easy
allergen Ara h 1, Anal. Chim. Acta 880 (2015) 52–59.
implementation of the developed methodology in analytical food [17] F. Conzuelo, M. Gamella, S. Campuzano, A.J. Reviejo, J.M. Pingarrón,
quality and safety laboratories performing routine analysis. More- Disposable amperometric magneto-immunosensor for direct detection of
over, it could also find application in inspection programs to enforce tetracyclines antibiotics residues in milk, Anal. Chim. Acta 737 (2012) 29–36.
[18] M. Pele, M. Brohée, E. Anklam, A. Van Hengel, Presence of peanut and
accurate labeling of commercial food products, or to monitor the hazelnut in cookies and chocolates: the relationship between analytical
effectiveness of cleaning processes from production units in the results and the declaration of food allergens on product labels, Food Addit.
food industry, thereby protecting both producers and consumers Contam. 24 (2007) 1334–1344.
[19] M. Gamella, S. Campuzano, F. Conzuelo, A.J. Reviejo, J.M. Pingarrón,
against peanut adulteration, misrepresentation and unintentional Amperometric magnetoimmunosensors for direct determination of D-dimer
ingestion of hidden allergens. in human serum, Electroanalysis 24 (2012) 2235–2243.
 V. Ruiz-Valdepeñas Montiel et al. / Sensors and Actuators B 236 (2016) 825–833 833

[20] B. Esteban-Fernández de Ávila, V. Escamilla-Gómez, S. Campuzano, M.

Pedrero, J.P. Salvador, M.P. Marco, J.M. Pingarrón, Ultrasensitive Rebeca Magnolia Torrente-Rodríguez received her
amperometric magnetoimmunosensor for human C-reactive protein degree in “Science and Chemical Technology” from the
quantification in serum, Sens. Actuators B: Chem. 188 (2013) 212–220. Universidad Complutense de Madrid (Spain) in July 2012.
[21] R.C. Alves, F.B. Pimentel, H.P.A. Nouws, W. Correr, M.B. González-García, From October 2012 to the present she is working on her
M.B.P.P. Oliveira, C. Delerue-Matos, Detection of the peanut allergen Ara h 6 in Ph.D. in Analytical Chemistry as a member of “Electroanal-
foodstuffs using a voltammetric biosensing approach, Anal. Bioanal. Chem. ysis and Electrochemical (Bio) sensors” research group in
407 (2015) 7157–7163. the same university. From April to August 2015 she joined
[22] T. Watanabe, H. Akiyama, S. Maleki, H. Yamakawa, K. Iijima, F. Yamazaki, T. the research group of Prof. Huangxian Ju at the State Key
Matsumoto, S. Futo, F. Arakawa, M. Watai, T. Maitani, A specific qualitative Laboratory of Analytical Chemistry for Life Science in Nan-
detection method for peanut (Arachis hypogaea) in foods using polymerase jing University (China) as a visiting student. Her main area
chain reaction, J. Food Biochem. 30 (2006) 215–233. of interest includes the design and development of novel
[23] E. Scaravelli, M. Brohee, R. Marchelli, A. Van Hengel, Development of three electrochemical biosensors for the detection of cancer
real-time PCR assays to detect peanut allergen residue in processed food biomarkers in biological samples as useful point-of-care
products, Eur. Food Res. Technol. 227 (2008) 857–869. devices.
[24] R. Köppel, V. Dvorak, F. Zimmerli, A. Breitenmoser, A. Eugster, H.U. Waiblinger,
Two tetraplex real-time PCR for the detection and quantification of DNA from
eight allergens in food, Eur. Food Res. Technol. 230 (2010) 367–374. A. Julio Reviejo received his Ph.D. in Chemical Sciences
(Analytical Chemistry) in 1991 from the Universidad Com-
plutense de Madrid. In 1992 he stayed at the New Mexico
Biographies State University for his postdoctoral training with Prof.
Joseph Wang. Since 1991, he has been working in the Ana-
lytical Chemistry Department of the Chemistry Faculty of
this University, where he is Professor of Analytical Chem-
Víctor Ruiz-Valdepeñas Montiel received his Degree in istry since 1988. He collaborates in the “Electroanalysis
Chemistry and his Master Degree in “Chemical Science and Electrochemical (Bio) sensors” research group. His
and Technology” from the Universidad Complutense de areas of interest include the development enzymatic and
Madrid (Spain) in June 2013 and 2014, respectively. He is immune- electrochemical sensors, molecular imprinted
working on his Ph.D. in Analytical Chemistry in the Uni- polymers, bioelectrocatalysis, and electrode modification.
versidad Complutense de Madrid (Spain) since September He is co-author of more than ninety research papers.
2014. He belongs to the “Electroanalysis and Electrochem-
ical (Bio) sensors” research group. His research interests
include the development of electrochemical biosensors Maria Stella Cosio received the master in Chemical and
for the determination of protein and genetic markers of Food Technology from the University of Parma (Italy)
importance for clinical diagnosis and food security. in 1996. Since 1991, she has been working in Food
Electroanalysis, Department of Food, Environmental and
Nutritional Sciences (DeFENS), University of Milan, where
she is associate professor of Food Analytical Chemistry
Alessandro Pellican received his degree in “Chemistry since 2003. Her area of interest and experience include
and Pharmaceutical Technology” from the University of the development of electrochemical sensors and biosen-
Messina in March 2012 and his Ph.D. in “Biotechnology sors, array of sensors (electronic nose and tongue), and
of Food” at Dept. of Food, Environmental and Nutritional chemically modified electrode. Moreover, she is trained
Science from the University of Milan in December 2015. in techniques such as FIA and HPLC with electrochemi-
He spent four months of his Ph.D. in the laboratory of Ana- cal detection as analytical tools for food quality and safety
lytical Chemistry, at Department of the Chemistry of the control.
Universidad Complutense de Madrid. His areas of inter-
est include the development of electrochemical sensors,
nanosensors, biosensors and immunosensors for determi- José M. Pingarrón obtained his Ph.D. in Sciences from
nation of biological and chemical compounds in food. Complutense University of Madrid. He is Professor of Ana-
lytical Chemistry at Complutense University of Madrid
and head of the group “Electroanalysis and electro-
Susana Campuzano received her Ph.D. in analytical chem- chemical (bio) sensors”. Current research includes the
istry from the Universidad Complutense de Madrid (Spain) development of nanostructured electrochemical biosen-
in 2004. She is Associate Professor at the Analytical sors, including enzyme electrodes, immunosensors and
Chemistry Department of the Chemistry Faculty of the genosensors for the ultrasensitive detection of bacteria,
Universidad Complutense de Madrid where she collab- low molecule weight hormones and cancer markers as
orates in the “Electroanalysis and Electrochemical (Bio) well as electrochemical arrays for multiplexed detection.
sensors” research group headed by Prof. J.M. Pingarrón. He is Associate Editor of the journal Electroanalysis and
She worked as a Research Scholar in the research group Vice-president of the Spanish Royal Society of Chemistry.
of Prof. J. Wang at the Department of Nanoengineering in He has authored over 300 research papers and several
UCSD (USA) from January 2010 to July 2011. Her areas of books and book chapters.
interest include the development of enzymatic, immuno
and DNA electrochemical sensors and advanced nanoma-
chines. She has authored over 90 research papers and
several book chapters.