Sensors and Actuators B 236 (2016) 825–833

Contents lists available at ScienceDirect

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
The widespread use of peanut products in food industry, the
severity of the symptoms even for trace quantities in highly sensi-
tized people [1], and the persistence in afflicted people have made
peanut-provoked allergy a major health concern. Although in most
people with peanut allergy, symptoms develop after ingesting less
than 1 peanut, which contains about 200 mg of protein [2], in highly
sensitized people, subjective symptoms were reported with doses
as low as 100 ␮g, and objective signs were evident with 2 mg [3,4].
Moreover, unlike other food-induced allergies, e.g., allergies trig-
gered by milk or egg proteins which mainly affect children and
∗ Corresponding authors. Fax: +34 913944329.
E-mail addresses: victor lega90@hotmail.com (V. Ruiz-Valdepeñas Montiel),
alessandro.pellicano@unimi.it (A. Pellicanò), susanacr@quim.ucm.es
(S. Campuzano), rebeca.magnolia@gmail.com (R.M. Torrente-Rodríguez),
reviejo@quim.ucm.es (Á.J. Reviejo), stella.cosio@unimi.it (M.S. Cosio),
pingarro@quim.ucm.es (J.M. Pingarrón).
1
These authors have contributed equally to this work.
disappear when growing up, this allergy is often a lifelong condi-
tion, the most common food-related cause of fatal allergic reactions
in Western countries [5] and its prevalence is increasing worldwide
[6,7].
To date, there is no cure for peanut allergy and no therapeu-
tic treatment is available to reduce the severity of this allergy.
Strict avoidance of peanuts and peanut-ingredients remains the
mainstay of management. However, full avoidance of peanuts is
very difficult because peanut seeds are currently widely used as
source of human food ingredients due to their high quality pro-
tein (22–30%) and oil content (44–56%) [8]. Moreover, the ingestion
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
for detection and quantification of peanut allergens are necessary
for ensuring the compliance of food labeling and for improving
consumer protection.
Over 13 allergenic components identified in peanuts, Ara h 2
and Ara h 6 are the best predictors of a severe allergic response

http://dx.doi.org/10.1016/j.snb.2016.01.123
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
contributes 5.9–9.3% to the total protein content of a peanut, has
been identified as the most offending peanut allergen [11] and as
an important predictor of clinical reactivity to peanut [6,8].
The detection of peanut allergens in food products is sometimes
a challenging task since they are often only present unintention-
ally and in trace amounts, or can be masked by compounds of
the constituting food matrix. Moreover, insufficient knowledge on
threshold levels (established by human oral challenge studies) is
available and, therefore, there is general agreement that the achiev-
able detection limits need to be as low as possible. Indeed, the
analytical community and especially standardization bodies are
looking for validated methods that can detect food allergens at
<10 mg kg−1 concentrations [12]. Currently, the main analytical
techniques used to detect peanut allergens can be classified into
protein-based or DNA-based assays. Protein-based assays detect
specific peanut allergen (Ara h 1 or Ara h 2), using enzyme-linked
immunosorbent assays (ELISAs), or total soluble peanut proteins.
DNA-based techniques detect the presence of allergens by ampli-
fying a specific DNA fragment of a peanut allergen gene through
polymerase chain reaction (PCR). However, false positive results
due to cross-reactivity with other nuts [9] as well as large differ-
ences in quantitative results obtained with available ELISA kits and
the high numbers of replicates for samples or an external stan-
dard required by all the described PCR methods have hindered their
application to processed foods or complex food matrices [13].
Electrochemical immunosensors have emerged as powerful
alternatives to the above mentioned techniques for the detection
and quantification of allergenic proteins. However their applica-
tions in this field are still scarce [14]. To the best of our knowledge,
no electrochemical immunosensor has been so far reported for the
determination of Ara h 2. This paper describes the first electrochem-
ical immunosensor for the selective and sensitive determination of
Ara h 2. The proposed design implies the use of functionalized mag-
netic beads (MBs) which have demonstrated to constitute powerful
tools to construct electrochemical immunosensor and minimize
sample matrix effects in the analysis of complex samples such as
food extracts [15,16]. In this particular case, the so called mag-
netoimmunosensor is based on a sandwich configuration, using a
matched-pair antibody set for a sandwich Ara h 2 immunoassay.
The capture antibody is immobilized onto carboxylic acid-modified
magnetic beads (HOOC-MBs) whereas the detector antibody was
labeled with a secondary HRP-conjugated anti-rabbit IgG (F(ab )2 -
HRP). The electrochemical detection of the affinity reactions was
carried out at disposable screen-printed carbon electrodes (SPCEs)
using hydroquinone (HQ) as electron transfer mediator and H2 O2
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
samples at trace level.
2. Experimental
2.1. Materials
Amperometric measurements were performed with a CHI812B
potentiostat (CH Instruments) controlled by software CHI812B;
screen-printed carbon electrodes (SPCEs) (DRP-110, DropSens),
consisting of a 4-mm diameter carbon working electrode, a car-
bon counter electrode and an Ag pseudo-reference electrode, were
employed as transducers and specific cable connector (ref. DRP-
CAC also from DropSens, S.L.) acted as interface between the SPCEs
and the potentiostat. All measurements were carried out at room
temperature.
A Bunsen AGT-9 Vortex was used for the homogenization of
the solutions. A Thermomixer MT100 constant temperature incu-
bator shaker (Universal Labortechnik) and a magnetic separator
Dynal MPC-S (product no. 120.20, Dynal Biotech ASA) were also
employed. Capture of the modified-MBs onto the SPCE surface was
controlled by a neodymium magnet (AIMAN GZ) embedded in a
homemade casing of Teflon. Centrifuges Cencom (J.P. Selecta S.A.)
and MPW-65R were used in the extraction steps.
All the reagents were of the highest available grade. Sodium
di-hydrogen phosphate (Cat. No.: SO03331000), di-sodium hydro-
gen phosphate (Cat. No.: SO03371000), Tris–HCl (Cat. No.:
TR04251000), NaCl (Cat. No.: SO02241000) and KCl (Cat. No.:
PO02001000) were purchased from Scharlab. Tween® 20 (Cat.
No.: 27,434-8), N-(3-dimethylaminopropyl)-N -ethylcarbodiimide
(EDC, Cat. No.: 171440100), N-hydroxysulfosuccinimide (sulfo-
NHS, Cat. No.: 438650010), ethanolamine (Cat. No.: E9508),
hydroquinone (HQ, Cat. No.: H-9003), hydrogen peroxide (H2 O2 ,
30%, w/v, Cat. No.: H1009) and albumin from chicken egg white
(OVA, Cat. No.: A-5503) were purchased from Sigma–Aldrich. 2-(N-
Morpholino)ethanesulfonic acid (MES, Cat. No.: 1080) and bovine
serum albumin (BSA Type VH, Cat. No.: 1066) were purchased from
Gerbu and commercial blocker casein solution (a ready-to-use, PBS
solution of 1% w/v purified casein) was purchased from Thermo
Scientific (Cat. No.: 37528). Carboxylic acid-modified MBs (HOOC-
MBs, 2.7 ␮m Ø, 10 mg mL−1 , Dynabeads® M-270 Carboxylic Acid,
Cat. No.: 14305D) were purchased from Dynal Biotech ASA. The cap-
ture antibody (Mouse monoclonal IgG1, 1C4, clone 1C4 G4 A9, AbC),
the detector antibody (Polyclonal rabbit antiserum raised against
natural purified Ara h 2, AbD), the Ara h 2 standard components
of the Ara h 2 ELISA Kit (Cat. No.: 1C4/AH2), used for comparison
purposes, and the Ara h 1 standard (Cat. No.: EL-AH1), were pur-
chased from Indoor Biotechnologies, Inc. Peroxidase-conjugated
AffiniPure F(ab )2 Fragment Goat anti-Rabbit IgG (F(ab )2 -HRP), Fc
Fragment Specific was purchased from Jackson Laboratories (Cat.
No.: 111-036-046).
All buffer solutions were prepared with water from Milli-
pore Milli-Q purification system (18.2 M cm). Phosphate-buffered
saline (PBS) consisting of 0.01 M phosphate buffer solution contain-
ing 137 mM NaCl and 2.7 mM KCl; 0.01 M sodium phosphate buffer
solution consisting of PBS with 0.05% Tween® 20 (pH 7.5, PBST);
0.05 M phosphate buffer, pH 6.0; 0.1 M phosphate buffer, pH 8.0;
0.025 M MES buffer, pH 5.0 and 0.1 M Tris–HCl buffer, pH 7.2. Acti-
vation of the HOOC-MBs was carried out with an EDC/sulfo–NHS
mixture solution (50 mg mL−1 each in MES buffer, pH 5.0). The
blocking step was accomplished with a 1 M ethanolamine solution
prepared in a 0.1 M phosphate buffer solution of pH 8.0.
2.2. Modification of MBs
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
stirring (950 rpm, 25 ◦ C). Between each step the particles were con-
centrated using a magnet and, after 4 min, the supernatant was
discarded. The carboxylic groups of MBs were activated by incuba-
tion during 35 min in 25 ␮L of the EDC/sulfo–NHS mixture solution.
The activated MBs were washed twice with 50 ␮L of MES buffer
and re-suspended in 25 ␮L of a 50 ␮g mL−1 antiAra h 2 solution in
MES buffer. The AbC was captured onto the activated beads at 25 ◦ C
under continuous stirring (950 rpm) during 15 min. Subsequently,
the AbC-modified MBs were washed twice with 50 ␮L of MES buffer
solution. Thereafter, the unreacted activated groups on the MBs
were blocked by adding 25 ␮L of the 1 M ethanolamine solution
in 0.1 M phosphate buffer, pH 8.0, and incubating the suspension
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
washing step with 50 ␮L of 0.1 M Tris–HCl buffer (pH 7.2) and two
more with 50 ␮L of the commercial blocker casein solution.
The immunoassay was carried out as follows: the AbC-coated
MBs were re-suspended in 25 ␮L of a mixture solution prepared in
commercial blocker casein solution containing the target Ara h 2
(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
of PBST buffer solution (pH 7.5), the modified MBs were incubated
with F(ab )2 -HRP (1/10,000) solution in PBST (pH 7.5) during 30 min
(950 rpm, 25 ◦ C). Two additional washings with 50 ␮L of PBST (pH
7.5) were carried out.
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.
The different procedures tested in the optimization of the num-
ber of steps involved in the immunoassay procedure were: (1) one
single step involving both Ara h 2 capture, sandwiching with AbD
and labeling with F(ab )2 -HRP by 30 min incubation of the AbC-MBs
in a mixture solution containing Ara h 2, AbD and F(ab )2 -HRP; (2A)
two steps involving 30 min incubation with the Ara h 2 solution, fol-
lowed by another 30 min incubation step in the mixture solution
containing AbD and F(ab )2 -HRP; (2B) two steps involving 30 min
incubation in a mixture solution containing Ara h 2 and AbD, fol-
lowed by 30 min incubation in the F(ab )2 -HRP solution; (3) three
steps implying sequential incubations of 30 min in Ara h 2, AbD and
F(ab )2 -HRP solutions, respectively.
The storage stability of the AbC-MBs was checked by keeping
them at 4 ◦ C in microcentrifuge tubes containing 50 ␮L of filtered
PBS. Two replicates of the stored conjugates were incubated each
working day in solutions containing 0 or 2.5 ng mL−1 Ara h 2 fol-
lowing the optimized protocol. A control chart was constructed by
setting as the central value the average current value calculated
from 10 measurements made the first day of the study for both Ara
h 2 concentration levels, while the upper and lower limits of control
were set at ±3 × SD of these initial values.
2.3. Amperometric measurements
To perform the amperometric measurements the SPCE was
positioned on a homemade casing of Teflon with neodymium
magnet encapsulated, and the 45 ␮L of the modified MBs suspen-
sion were magnetically captured on the SPCE surface according
to the procedure described earlier [17]. Then, the magnet hold-
ing block was immersed into an electrochemical cell containing
10 mL of 0.05 M phosphate buffer of pH 6.0 and 1.0 mM HQ (pre-
pared just before performing the electrochemical measurement).
Amperometric measurements in stirred solutions were made by
applying a detection potential of −0.20 V vs Ag pseudo-reference
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-
metric signals given through the manuscript corresponded to the
difference between the steady-state and the background currents.
2.4. Analysis of real samples
The Ara h 2 amperometric magnetoimmunosensor was applied
to the analysis of different food samples containing unknown
amounts of endogenous Ara h 2 and also samples free of peanuts
(wheat flour) spiked at trace levels.
Different types of foodstuffs, purchased in local supermarkets,
were analyzed: wheat flour, hazelnuts; peanuts (peanut flour, raw,
fried and chocolate-coated); chocolate bars with roasted peanuts,
nougat, caramel and milk chocolate; multicereals bars with up to
59% (w/w) of whole roasted peanuts; chocolate chip cookies with
caramelized almonds; peanut creams and peanut oil.
827
Table 1
Optimization of the different experimental variables affecting the performance of
the amperometric magnetoimmunosensor for Ara h 2 (by comparison of the S/B
ratio obtained in the presence of 5.0 and 0.0 ng mL−1 Ara h 2). See text for other
used variables.
Variable Tested range Selected value
[AbC], ␮g mL−1 2.5–100 50
tAbC , min 15–90 15
[AbD], dilution 1/250–1/5000 1/1000
[F(ab )2 -HRP], dilution 1/250–1/50,000 1/10,000
tAg + AbD , min 15–90 45
tF(ab ) 2-HRP , min 15–90 30
Regarding the analysis of spiked samples, peanut-free wheat
flour (verified using a commercial Ara h 2 ELISA spectrophotometric
kit) was spiked with different amounts of peanut flour that con-
sisted of 100% raw peanut (unknown variety) from a commercial
retailer (Frinuts). Accordingly, a series of mixtures containing 0.05,
0.025, 0.01, 0.0075, 0.005, 0.001, 0.0005 and 0.0001% w/w of peanut
were prepared.
The following protocol was used for the extraction of pro-
teins present in peanuts in all the food samples analyzed: 0.5 g
of accurately weighted ground sample (previously blended) were
introduced in plastic tubes and incubated in 5.0 mL of Tris–HCl (pH
8.2) overnight at 60 ◦ C under continuous stirring (950 rpm). Regard-
ing chocolate samples, they were frozen at −20 ◦ C before blending,
and 0.5 g of skimmed milk powder (Central Lechera Asturiana® )
were added during the extraction in order to avoid masking of the
target protein by tannins [12]. Subsequently the aqueous phase
was isolated by centrifugation involving a first step at 3,600 rpm
during 10 min and a second step at 10,000 rpm during 3 min (4 ◦ C)
for a 1-mL aliquot of the first supernatant [5,16,18]. The resulting
supernatant appropriately diluted was used to perform the Ara h 2
determination with the magnetoimmunosensor.
In order to make comparison, the same food extracts were also
analyzed by applying an ELISA method involving the use of the same
immunoreagents.
3. Results and discussion
The fundamentals of the magnetoimmunosensor configuration
as well as of the electrochemical transduction used in this work are
displayed in Fig. 1. As can be observed, all the immunoreactions
involved in the assay occurred on the MBs surface. After magnetic
capturing of the MBs bearing the sandwich-type immunoconju-
gates on the working electrode surface, amperometric detection
of the HRP enzymatically catalyzed reduction current generated in
the presence of the HQ/H2 O2 redox system was carried out.
3.1. Optimization of experimental variables
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
and a commercial blocker casein solution was employed. This
blocking solution has proven to be highly effective for minimiza-
tion of non-specific adsorptions [16]. Therefore, the sandwich
configuration approach was feasible for the target protein determi-
nation. Subsequently, the optimization of experimental variables
involved in the magnetoimmunosensor preparation and function-
ing was accomplished. The adopted criterion of selection for each
variable was the magnitude of the ratio between the amperomet-
ric responses measured at −0.20 V in the presence (signal, S) of
5.0 ng mL−1 Ara h 2 and in the absence of target protein (blank, B)
(S/B ratio). All the tested variables, the ranges into which they were
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
per assay were optimized in previous works [19,20].
In addition to the experimental variables collected in Table 1
and with the aim of simplifying as much as possible the whole
protocol and reducing the assay time, the effect of the number of
involved working steps in the immunoassay procedure following
the protocol described in Section 2.2 was evaluated. The obtained
results indicating clearly as the protocol involving an initial incu-
bation step with the mixture solution of the target protein and the
AbD, and a latter incubation with F(ab )2 -HRP (bars 2B in Fig. 2)
provided the largest S/B current ratio. This effect was most likely
due to the lower target recognition efficiency as a consequence
of increased steric hindrance when all the immunoreagents were
mixed in homogeneous solution (bars 1) or the AbD was incubated
together with the F(ab )2 -HRP (bars 2A). It is worth to mention also
that the S/B ratio was significantly larger when the target protein
was incubated with the AbD in the same step (bars 2B) than in the
case of applying separate steps (bars 3), probably due to a more
efficient binding with AbD when Ara h 2 is free in solution. Accord-
ingly, the protocol 2B was employed for the implementation of the
magnetoimmunosensor.
3.2. Analytical characteristics
A calibration plot was constructed with Ara h 2 standards under
the selected experimental conditions. As it can be seen in Fig. 3, a
linear relationship (r = 0.999) between the measured current and
the Ara h 2 concentration was found over the 87–10,000 pg mL−1
range, with a slope and intercept values of (198 ± 2) nA mL ng−1 and
(87 ± 8) nA, respectively. The LOD and the determination limit (LQ),
26 and 87 pg mL−1 , respectively, were calculated according to the
3s m−1 and 10s m−1 criteria, respectively, where m is the slope of
the calibration plot and s was estimated as the standard deviation
of ten amperometric signals obtained without target Ara h 2.
Amperometric measurements for 2.5 ng mL−1 Ara h 2 carried out
with 10 different magnetoimmunosensors prepared using the opti-
mized protocol yielded a relative standard deviation (RSD) value of
3.3%, thus indicating a great reproducibility of the whole magne-
toimmunosensor fabrication and the signal transduction protocols.
The results obtained in the storage stability study of the AbC-
MBs (not shown, see additional details in Section 2.2) showed
that the magnetoimmunosensors response remained within the
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

The selectivity of the magnetoimmunosensor was evaluated
both toward Ara h 1, the other major allergen in peanuts, and
non-target proteins such as BSA and OVA which can coexist with
the target protein in food extracts. These tests were performed
by comparing the current values measured with the magnetoim-
munosensor for 0 and 2.5 ng mL−1 Ara h 2 in the absence and
in the presence of these potential interfering compounds. Fig. 4
clearly shows as no significant interference was apparent for Ara h
2 determination as a result of the presence of the three tested non-
target proteins even at the large concentrations assayed for BSA and
OVA. It is worth to highlight that no noticeable cross-reactivity was
3.4. Determination of Ara h 2 in food samples
The real usefulness of the developed methodology was eval-
uated by determining the target allergen protein in different food
extracts containing variable unknown content of endogenous Ara h
2 as well as in spiked target protein-free samples. The used protocol
is illustrated in Fig. 5.
No statistically significant differences were observed between
the slope value of the calibration plot constructed with Ara h 2
standards and the slope values of the calibration graphs recorded
from all the prepared extracts once they were adequately diluted
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
concluded that no significant matrix effects were apparent once
the sample dilution factors required to fit the target analyte con-
centration into the linear range of the calibration graph shown in
Fig. 3, were applied, confirming also the effectiveness in the use
of MBs and blocker casein solution to minimize the non-specific
adsorption of components from complex samples. Therefore, Ara h
2 quantification could be accomplished by simple interpolation of
the measured current from the diluted samples into the calibration
plot constructed with Ara h 2 standard solutions.
The results obtained for all the assayed food extracts together
with the dilution factor applied to each analyzed extract are pre-
sented in Table 2. In addition, the obtained results were compared
with those provided by a commercial ELISA kit using the same
immunoreagents. An excellent correlation was found between the
Ara h 2 concentrations measured with the ELISA kit and those
obtained with the amperometric magnetoimmunosensor (Fig. 5b)
with the confidence intervals (at a significance level of ˛ = 0.05)
for the slope and intercept including the unit and the zero val-
ues, respectively. However, the use of the magnetoimmunosensor
allowed the analysis to be made in half-time compared with
the ELISA method (2 vs 4 h once the AbC-MBs and AbC-plate
were prepared, respectively), simplified largely the whole analyt-
ical procedure required smaller sample and bioreagents volumes
(25 vs 100 ␮L), with the consequent saving in cost per analy-
sis, and can be easily automated and implemented with portable
and cost-effective instrumentation. These characteristics make
the magnetoimmunosensor an attractive and user-friendly tool
to monitor routinely the food quality and to perform decentral-
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
products [9].
Regarding the analysis of wheat flour containing no detectable
content of the target allergen (see Table 2), it was spiked with dif-
ferent increasing amounts of peanut flour and the corresponding
extracts were prepared and analyzed as described in Section 2.4.
The results are summarized in Fig. 6, where the measurements
made with an Ara h 1 immunosensor previously developed in our
group are also displayed [16]. These results demonstrated that the
Ara h 2 magnetoimmunosensor was able to detect clearly 0.0005%
(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%
[14,21]. This highly improved sensitivity for peanut detection can
be attributable to the use of different immunoreagents and label-
ing protocol (use of F(ab )2 -HRP instead of Strep-HRP) and to a
more efficient extraction of the smaller target protein (17.5 vs
65 kDa for Ara h 1) from the food samples. This enhanced sensitiv-
ity represent a major comparative advantage taking into account
the serious public health problem that the degree of contamina-
tion of commercial food samples with peanuts, whether fraudulent
or accidental, can cause in sensitive individuals. Moreover, it is
important to emphasize that although a level of 10 mg kg−1 is con-
sidered relevant for the detection of potentially hazardous residues
of undeclared allergens in foods, the achievement of lower detec-
tion limits, as it is the case of this work, may be highly helpful since
minimal amounts of the target allergen can be critical [9]. In fact,
in most people suffering peanut allergy, symptoms are developed
after ingesting less than 1 peanut and, in highly sensitized people,
subjective symptoms have been reported even with doses as low
as 100 ␮g [3,4].
Moreover, it is important to mention that the detection level
for peanuts achieved with the magnetoimmunosensor, about
5.0 mg kg−1 , incurred in wheat flour is in the range of some
of the most promising PCR-based approaches reported for sen-
sitive peanut determination, which are in the 2–10 mg kg−1
range [9,22–24]. Most interestingly, the LOD provided by the
immunosensor is achieved without performing any amplification
step.
4. Conclusions
A disposable amperometric magnetoimmunosensor for the
high sensitive determination of the allergenic protein Ara h 2 is
described for the first time in this work. This magnetoimmunosen-
sor allows a LOD of 26 pg mL−1 to be obtained for the target protein
and has clearly demonstrated successful applicability for the accu-
rate determination of the endogenous content of Ara h 2 in different
food extracts. The achieved results benchmarked against a com-
mercially available ELISA kit and allowed the detection of peanut
trace contents (5 mg kg−1 ) in spiked wheat flour samples. The great
analytical performance achieved also in terms of high selectivity,
accuracy, reliability and reproducibility in complex food matri-
ces, together with the flexibility and simplicity of the disposable
magnetoimmunosensor, mean important advantages for an easy
implementation of the developed methodology in analytical food
quality and safety laboratories performing routine analysis. More-
over, it could also find application in inspection programs to enforce
accurate labeling of commercial food products, or to monitor the
effectiveness of cleaning processes from production units in the
food industry, thereby protecting both producers and consumers
against peanut adulteration, misrepresentation and unintentional
ingestion of hidden allergens.
Acknowledgments
The financial support of the Spanish Ministerio de Economía
y Competitividad Research Projects, CTQ2015-64402-C2-1-R, and
the NANOAVANSENS Program from the Comunidad de Madrid
(S2013/MT-3029) are gratefully acknowledged. Financial support
by COFIN 2010–2011 (MIUR, 2010 AXENJ8 002) is also acknowl-
edged. R.M. Torrente-Rodríguez acknowledges a CPFD predoctoral
contract from the Spanish Ministerio de Economía y Competitivi-
dad.
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Biographies
Víctor Ruiz-Valdepeñas Montiel received his Degree in
Chemistry and his Master Degree in “Chemical Science
and Technology” from the Universidad Complutense de
Madrid (Spain) in June 2013 and 2014, respectively. He is
working on his Ph.D. in Analytical Chemistry in the Uni-
versidad Complutense de Madrid (Spain) since September
2014. He belongs to the “Electroanalysis and Electrochem-
ical (Bio) sensors” research group. His research interests
include the development of electrochemical biosensors
for the determination of protein and genetic markers of
importance for clinical diagnosis and food security.
Alessandro Pellican received his degree in “Chemistry
and Pharmaceutical Technology” from the University of
Messina in March 2012 and his Ph.D. in “Biotechnology
of Food” at Dept. of Food, Environmental and Nutritional
Science from the University of Milan in December 2015.
He spent four months of his Ph.D. in the laboratory of Ana-
lytical Chemistry, at Department of the Chemistry of the
Universidad Complutense de Madrid. His areas of inter-
est include the development of electrochemical sensors,
nanosensors, biosensors and immunosensors for determi-
nation of biological and chemical compounds in food.
Susana Campuzano received her Ph.D. in analytical chem-
istry from the Universidad Complutense de Madrid (Spain)
in 2004. She is Associate Professor at the Analytical
Chemistry Department of the Chemistry Faculty of the
Universidad Complutense de Madrid where she collab-
orates in the “Electroanalysis and Electrochemical (Bio)
sensors” research group headed by Prof. J.M. Pingarrón.
She worked as a Research Scholar in the research group
of Prof. J. Wang at the Department of Nanoengineering in
UCSD (USA) from January 2010 to July 2011. Her areas of
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.
833
Rebeca Magnolia Torrente-Rodríguez received her
degree in “Science and Chemical Technology” from the
Universidad Complutense de Madrid (Spain) in July 2012.
From October 2012 to the present she is working on her
Ph.D. in Analytical Chemistry as a member of “Electroanal-
ysis and Electrochemical (Bio) sensors” research group in
the same university. From April to August 2015 she joined
the research group of Prof. Huangxian Ju at the State Key
Laboratory of Analytical Chemistry for Life Science in Nan-
jing University (China) as a visiting student. Her main area
of interest includes the design and development of novel
electrochemical biosensors for the detection of cancer
biomarkers in biological samples as useful point-of-care
devices.
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
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-
istry since 1988. He collaborates in the “Electroanalysis
and Electrochemical (Bio) sensors” research group. His
areas of interest include the development enzymatic and
immune- electrochemical sensors, molecular imprinted
polymers, bioelectrocatalysis, and electrode modification.
He is co-author of more than ninety research papers.
Maria Stella Cosio received the master in Chemical and
Food Technology from the University of Parma (Italy)
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
since 2003. Her area of interest and experience include
the development of electrochemical sensors and biosen-
sors, array of sensors (electronic nose and tongue), and
chemically modified electrode. Moreover, she is trained
in techniques such as FIA and HPLC with electrochemi-
cal detection as analytical tools for food quality and safety
control.
José M. Pingarrón obtained his Ph.D. in Sciences from
Complutense University of Madrid. He is Professor of Ana-
lytical Chemistry at Complutense University of Madrid
and head of the group “Electroanalysis and electro-
chemical (bio) sensors”. Current research includes the
development of nanostructured electrochemical biosen-
sors, including enzyme electrodes, immunosensors and
genosensors for the ultrasensitive detection of bacteria,
low molecule weight hormones and cancer markers as
well as electrochemical arrays for multiplexed detection.
He is Associate Editor of the journal Electroanalysis and
Vice-president of the Spanish Royal Society of Chemistry.
He has authored over 300 research papers and several
books and book chapters.