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Advances in biosensor development
based on integrating
nanotechnology and applied to
food-allergen management
Rosa Pilolli, Linda Monaci, Angelo Visconti
The risks associated with the presence of hidden allergens in the food chain have raised the need for fast, sensitive, and reliable
methods to trace food allergens in different commodities.
We highlight advances and future trends in biosensor systems applied to food-allergen management. We discuss critical
aspects of biosensor development with particular emphasis on integrating nanotechnology.
ª2013 Elsevier Ltd. All rights reserved.
Keywords: Allergen; Biosensor; Electrochemical biosensor; Electromechanical biosensor; Electrochemical impedance spectroscopy (EIS); Food;
Nanomaterial; Nanotechnology; Optical detection; Surface-plasmon resonance (SPR)
Abbreviations: Ab, Antibody; CCD, Charge-coupled device; CHOM, Chicken ovomucoid; CL-larray, Chemiluminescence-based microarray; DNA,
Deossiribonucleic acid; DPV, Differential pulse voltammetry; DVD, Digital versatile disk; EIS, Electrochemical-impedance spectroscopy; ELISA,
Enzyme-linked immunosorbent assay; EU, European Union; f-EIS, Faradaic electrochemical-impedance spectroscopy; FPW-MEMS, Flexural plate-
wave micro-electromechanical systems; Fluo-larray, Fluorescence microarray; F-SLW, Front-surface long-wavelength; GCE, Glassy-carbon
electrode; IgE, IgG, IgY, Immunoglobulin E, G, Y; IMS, Immunomagnetic separation; LS, Light scattering; NA, Not available; nf-EIS, Non-Faradaic
electrochemical-impedance spectroscopy; NP, Nanoparticle; NB, Nile Blue; OVA, Ovalbumin; PCR, Polymerase chain reaction; P-L-Arg/MWCNT,
Poly(l-arginine)/multi-walled carbon nanotube; QCM, Quartz-crystal microbalance; QD, Quantum dot; RT-CA, Real-time chronoamperometry;
SERS, Surface-enhanced Raman scattering; SPR, Surface-plasmon resonance; SWV, Square-wave voltammetry
1. Introduction
Food allergy is nowadays regarded as a
problem of public-health relevance, the
main concern being the unintentional
exposure of allergic consumers to the
offending ingredient through allergen-
containing food. Even a little intake of
allergen can trigger unpredictable, highly
variable reactions, depending on the dose
and the sensitivity of affected individual,
thus compelling the allergic consumer to
avoid allergen-containing food totally.
The European Community fixed labeling
regulations for 14 allergenic food ingredi-
ents so it is mandatory for them to be la-
beled on the relevant food products [1].
Given the lack of official regulatory
thresholds, allergen-labeling laws have
taken a conservative approach to allergy-
risk management, so that no level of the
major food allergens can be deemed safe
for consumers and the presence of any
amount intentionally added to food must
be labeled. However, these requirements
only affect allergens used as ingredients,
and the potential for undeclared allergens
in foods is not addressed by the directive.
The presence of an allergen can also be
caused by cross-contamination in a shared
production line or the raw-material or
ingredient supply chains [2].
Both unlabeled contaminated products
and labeled products with negligible con-
tamination must be avoided, so precau-
tionary labels should be used only where
cross-contact is likely to occur and health
risks are expected. In order to identify when
such a situation applies, the concepts of
assessing and managing the ‘‘risk’’ are ap-
plied to allergen contamination [3]. The
probabilistic modeling of risk assessment,
Rosa Pilolli,
Linda Monaci*,
Angelo Visconti
Institute of Sciences of Food
Production,
National Research Council of
Italy (ISPA-CNR),
Via Amendola 122/O,
70126 Bari, Italy
*
Corresponding author.
Tel.: +39 0805929343;
Fax: +39 0805929374;
E-mail: linda.monaci@
ispa.cnr.it
Trends Trends in Analytical Chemistry, Vol. 47, No. , 2013
12 0165-9936/$ - see front matter ª2013 Elsevier Ltd. All rights reserved. doi:http://dx.doi.org/10.1016/j.trac.2013.02.005
first described by Spanjersberg et al. [4] in 2007, is now-
adays considered to be the most promising approach. This
methodology allows an estimation of the allergic popu-
lation percentage that may present a reaction due to the
presence of a certain level of undesired allergen in a food
product. This, evidently, asks the food manufacturers for a
sound knowledge about the frequency and the extent of
allergen cross-contamination during production and en-
tails the critical choice of threshold-decision level. A risk of
0% would be only possible when the industry is able to
produce food ‘‘free of allergens’’, but such an option is not
cost effective for the consumer or the manufacturer [5].
Risk management focuses mainly on enforcement of good
manufacturing practices (i.e. allergen clean-up and con-
trol measures). However, although allergen clean-up of
shared processing lines has been identified as one of the
critical points for effective allergen control, so far few
investigations have focused on this topic. It is only re-
cently that a certain amount of effort was put into eval-
uating the best practices at the industrial scale for allergen
sanitization in order to reduce the undesired cross-con-
tamination down to a threshold level at which it becomes
irrelevant for public health [6,7]. Clearly, two main par-
allel research directions need to be followed:
clinical investigations for the identification of relevant
threshold levels; and,
technological advances for validation and harmoniza-
tion of analytical methods.
From the analytical point of view, the extreme sensi-
tivity of allergens or their specific markers to various
processing and matrix effects, and the consequent issues
occurring in identifying reference materials hamper the
development and the harmonization of reliable reference
protocols. To fix some of these open issues, the effect of
food processing on allergens is being considered more
and more frequently, with particular emphasis on how
such processing can modify the detection performance.
Complementary to confirmatory techniques {e.g., li-
quid chromatography mass spectrometry (LC-MS)
[8,9]}, rapid diagnostic tools are increasingly being
promoted for food companies to verify the efficiency of
their management schemes for food safety [10].
As a result of a recent survey carried out within
European Union (EU) networked project MoniQA [11],it
was definitely established that the food industry is rap-
idly extending the range of rapid tests utilized, with
particular interest in implementation of allergen-related
test kits, ranked second in requests after microbiological
tests [12]. Indeed, in food allergens, among other areas,
rapid-test methods can provide a valuable tool for vali-
dating and verifying the effectiveness of sanitation
practices, consequently minimizing the risk of cross-
contact contamination.
The most commonly used rapid methods for routine
monitoring are enzyme-linked immunosorbent assays
(ELISAs) in 96-well-plate format. Even though some
level of automation has been achieved in the recent
years, ELISAs remain laborious, time consuming and
expensive, particularly when multiple targets need to be
screened.
Biosensors represent a potential alternative to ELISAs,
and provide probably one of the most promising ways to
solve some problems concerning simple, fast, reproduc-
ible, and cheap multi-analyte detection. A biosensor is an
integrated receptor-transducer device, which converts the
biological-recognition event into a measurable chemical-
physical signal, which is proportional to the target con-
centration. The receptor can be an antibody raised against
an allergen, a single-stranded DNA molecule capable of
hybridizing with an allergen-specific DNA fragment, or an
aptamer selected to recognize the target allergen directly.
Featuring high speed of execution, ease of use and
high degree of automation, biosensors have all the po-
tential for direct, real-time, on-line, monitoring of aller-
gens along the production chain, and there remain
important challenges (i.e. the dependence of the effi-
ciency of allergen detection on matrixes and processing),
both strictly related to the specific target and signifi-
cantly affecting any biosensor performance. However, in
recent years, great efforts were devoted to this innovative
application field for biosensors (see Fig. 1).
In particular, taking advantage of advances in mate-
rials science, new opportunities of improvement of the
current technology were experimented on, by imple-
menting nanomaterials. The integration of the high
specificity of biological receptors with the unique optical,
electrical and electrochemical properties of the nanom-
aterials provides novel interesting alternatives to con-
ventional platforms, by exceeding the sensitivity of
existing techniques [13,14].
The present article discusses several critical aspects of
biosensor development for food-allergen management.
We review the literature of the past four years using a
transduction-based classification (optical, electrochemi-
cal and electromechanical), which highlights the most
important achievements and the new research trends,
with particular emphasis on the potential for imple-
menting nanotechnology.
2. Optical biosensors
Optical biosensors represent powerful detection tools with
applications in food, healthcare, and environmental
monitoring [15]. Combining the selectivity of biology with
the processing power of modern microelectronics and
optoelectronics, they offer great analytical potential with
major applications in food safety, thanks to advantages of
detecting analytes in complex matrices with minimal
sample treatment. Most optical biosensors are based upon
measurement of changes in the surface properties of a
sensor chip when the analyte is bound to a sensing layer
Trends in Analytical Chemistry, Vol. 47, No. , 2013 Trends
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on the device by sorption or complex formation of the
receptor-target couple.
Various chemical-physical phenomena are exploited
for different optical biosensors, including absorption,
fluorescence and surface-plasmon resonance (SPR).
UV/visible absorption spectroscopy is a well-estab-
lished technique in macroscale analytical chemistry.
This approach is typically exploited in colorimetric as-
says for qualitative and quantitative determination of
food contaminants. In most cases, changes in optical
density or color are sufficient for diagnosis, so instru-
mentation for absorbance measurements tends to be
much simpler than that needed for other methods.
Fluorescence is the most widely-used method of optical
detection due to the well-established, highly sensitive,
selective labeling techniques and the number of available
fluorophores borrowed from conventional genomic and
proteomic analysis. Fluorescent detection has several
advantages over other techniques, but fluorescent dyes
remain rather costly, have a limited shelf life, and are
often influenced by chemical factors (e.g., pH), which
may vary from sample to sample.
SPR biosensors are optical sensors exploiting the
refractive-index change close to a sensor surface. When
light is incident on a thin metal film, at a specific angle,
through a prism, it interacts with the metal free elec-
trons (surface plasmons) and generates an evanescent
wave. At this angle, the intensity of reflected light de-
creases sharply. The angle at which this reduction oc-
curs is called the SPR angle (h), and it varies with the
refractive index of the dielectric medium (usually buffer)
on the opposite side of the metal film (usually gold).
When the biomolecular recognition element is immobi-
lized on the gold surface, SPR angle hchanges as a result
of the interaction between the immobilized receptor and
the analyte in solution (see Fig. 2a). SPR angle his
measured by rotating a narrowly-focused laser beam and
looking for a reflectance minimum (conventional SPR)
or by using a slightly divergent laser beam and imaging
the reflectance angle spectrum exiting the prism [imag-
ing SPR (i-SPR)].
2.1. Surface-plasmon-resonance-based detection
Boasting high surface sensitivity, and real-time and la-
bel-free response, the SPR phenomenon has been widely
employed in different transduction configurations to
characterize biomolecular interactions. SPR allows us to
monitor various processes, i.e. DNA hybridization and
antibody–antigen binding [15] and DNA–protein inter-
actions [16].
Various assay formats are available (Fig. 2b–e). For
protein detection, usually, direct analyte binding to
surface-immobilized ligands on the sensor chip is per-
formed (see Fig. 2b), eventually enhancing the specificity
and the limit of detection (LOD) by using sandwich for-
mats, in which a second binder to the analyte is injected
(see Fig. 2c).
Recently, nanomaterials were implemented in the
biological-recognition element to amplify the SPR-signal
change. Magnetic nanoparticles (MNPs), particularly,
have been exploited to enhance detection sensitivity in
sandwich assays by increasing the mass change upon
specific binding at the near-surface sampled region.
Alternatively, gold nanomaterials, in addition to the
mass-change enhancement, provide an electronic cou-
pling interaction between intrinsically-localized SPR and
the thin-film surface plasmon, thus further improving
the overall sensitivity of the test [17].
We previously discussed the very first examples of
SPR-based biosensor for food-allergen detection in a re-
view paper [18]. Following those results, several papers
with specific investigation aims were published.
A label-free immunosensor was developed by Indyk for
the quantitative detection of a-lactalbumin in bovine
Figure 1. Trend of publications focused on biosensors featuring application in food allergen management, and nanotechnology integration, as
obtained from the Web of Science database (specific keywords: ‘‘biosens
*
’’, ‘‘allerg
*
’’ and ‘‘nano
*
’’).
Trends Trends in Analytical Chemistry, Vol. 47, No. , 2013
14 http://www.elsevier.com/locate/trac
milk [19]. The analytical interest in this particular milk
component arose from its potential as a marker of heat
treatment and its high nutritional value in supple-
menting infant formula milk. The technique was applied
to detection at endogenous levels in milk and colostrum
and at supplemental levels in infant formula milk and
whey-protein isolates (see Table 1).
Similarly, a fast immunoglobulin G (IgG) SPR immu-
nosensor was reported by Rossi and co-workers [20] for
the detection of colostrum adulteration in bovine and
caprine milk. The significantly short analysis time
achieved (<4 min) made this biosensor compatible with
the real-time monitoring of the milking of small rumi-
nants and with systematic control of large numbers of
samples in order to avoid negative economical and
technological consequences for dairy industry.
Another example of a biosensor for milk-adulteration
monitoring was provided by He et al. [21], who com-
bined immunomagnetic separation (IMS) and surface-
enhanced Raman scattering (SERS) to detect ovalbumin
(OVA), an egg-white protein added to whole milk.
Magnetic micro-beads were used to capture OVA from
milk first, then two SERS methods, solution-based and
substrate-based, were applied to analyze the IMS eluate:
solution SERS was assessed to be more capable of
quantitative analysis; and,
substrate SERS was assessed to be more sensitive for
qualitative analysis.
Figure 2. (A) Operating principle of surface-plasmon resonance (SPR) detection, and of the various immunoassay formats available: (B) direct; (C)
sandwich; (D) competitive; and, (E) inhibition.
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Table 1. Optical biosensors for detection of food allergens
Transduction Analyte Receptor Assay Matrixes Run time Nanomaterial LOD Ref.
SPR (direct label-
free detection)
a-lactalbumin Polyclonal-Ab Direct Milk, colostrum,
whey protein
concentrates,
infant formulae
– None 0.94 ng/mL [19]
SPR (direct label-
free detection)
Bovine Ig G
caprine IgG
Polyclonal-Ab Direct Milk <4 min None 2.6 ng/mL
3.1 ng/mL
[20]
i-SPR (direct label-
free detection)
Peanut, milk,
lupine, soy, egg,
hazelnut, almond,
cashew nut, brazil
nut, pine nut,
pecan, macadamia
nut, pistachio nut
Monoclonal and
polyclonal Ab
Direct Allergenic food
cookies
dark chocolate
8 min None low mg/kg range
for cookies and
chocolate
[26]
SPR (direct label-
free detection)
a-lactalbumin
b-lactoglobulin
BSA
Lactoferrin
IgG
Affinity-purified
polyclonal-Ab
Direct Raw and processed
milk whey fractions
milk-derived
products
NA None NA [25]
Optical fiber SPR
(direct label-free
detection)
Ara h1 Polyclonal Ab Sandwich Chocolate 20 min Magnetic
nanobead
0.09 lg/mL [22]
IMS-SERS Ovalbumin Polyclonal Ab Direct Whole milk 20 min Magnetic
microbeads + silver
dendrites
1lg/mL [21]
Colorimetric
(indirect detection,
enzyme label)
Lectin, Ara h3,
Gliadin, Ana o3,
Mitochondrion
DNA, 16S rRNA
DNA Sandwich Soybean, cashew,
peanut, wheat,
beef, chicken, fish,
shrimp, cereal bar,
chocolate chips,
wheat biscuit, dark
chocolate, fried
mud carps
30 min (after PCR) None Cashew: 0.5 pg [36]
Colorimetric
(indirect detection,
enzyme label)
Sin a1, ITS(18S-
26S), Jug r2,
Oleosin, Mtd, Pru
du1, Avenin, 2S
Albumin mRNA
DNA Sandwich Walnut, almond,
hazelnut, seed,
sesame, celery, oat,
mustard, lupine,
wheat, biscuits,
chocolate, omelet,
wafers, bread sticks
30 min (after PCR) None Sesame 0.5 pg [37]
Optical density
variation (indirect
detection, enzyme
label)
Cor a1, Ar h2, Le DNA Sandwich Seeds, cookies,
cereals,
chocolates, pastas,
soups, and snacks,
dairy products,
jams, ice cream,
frozen ready meals
2.5h incubation time None 1 lg/g [38]
F-SLW fluorimetry
(indirect detection,
NPs label)
Soy protein Polyclonal Ab Competitive Soy milk, soy
yoghourt, fruit, soy
juice
incubation time: 2 h ± 1.5 h Nile blue-doped
silica NPs (257 nm)
0.05 mg/L [39]
Abbreviations: SPR, Surface-plasmon resonance; IMS-SERS, Immuno-magnetic separation – surface-enhanced raman spectroscopy; F-SLW, Front-surface long-wavelength; Ab, Antibody; NP,
Nanoparticle; NA, Not available.
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Figure 3. (A) The different immunoassay strategies for a fiber-optic SPR biosensor. (B) On-chip direct allergen screening using imaging surface-
plasmon resonance (i-SPR): hydrogel-coated chip is spotted with allergen-specific antibodies using a continuous-flow microfluidic (CFM) spotter.
The sample is delivered to the chip using a flow cell (scale bar, 1 cm). (C) SPR image of the chip with anti-peanut (Pea), anti-pine nut (Pin), anti-
almond (Alm), anti-j-casein (j-Ca), anti-macadamia (Mac), anti-hazelnut (Haz), anti-pecan (Pec), anti-brazil nut (Bra), anti-lupine (Lup), anti-pis-
tachio nut (Pis), anti-cashew nut (Cas), anti-egg (Egg), anti-soy (Soy), and buffer (ref). Spot dimensions are 400 lm·600 lm. {Panels c and d
reprinted with permission from [26],ª2010 American Chemical Society}.
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Table 2. Biosensors for epitopes characterization and allergy diagnosis
Transduction Specific
allergen
Investigation aim Nanomaterial Ref.
SPR PNA lectin Characterization of IgE-binding epitopes
of peanut (Arachis hypogaea) PNA lectin
None [28]
SPR Caseins Determining topography of the casein
micelle surface
None [27]
SPR – Characterization of the aptamers – human
IgE interaction
None [30]
SPR b-conglutin
from lupine
Selection of aptamers for b-conglutin None [29]
SPR – Proof of concept of aptamer-
nanoparticle-based enhancement of
human IgE detection
Gold nanoparticles [31]
SPR – Proof of concept of nanorod-based
enhancement of human IgE detection
Gold nanorods [32]
SPR – Proof of concept of nanoparticles based-
enhancement of human IgE detection
Gold nanoparticles [33]
LS Peanut Ara h1 Screening of whole blood samples at
point-of-care for allergy diagnosis
Gold nanoparticles [34]
LS Peanut Ara h1 Screening of whole blood samples by
immunokinetic assay
Gold nanoparticles [35]
Dual
interferometry &
Fluo-larray
IgG,
b-lactoglobulin
Ara h1 and Phl
p1
Development and validation of
multiplexed method to calibrate and
quantify fluorescence signal for allergen-
specific IgE
None [40]
Fluo-larray Ovalbumin
ovomucoid
Conalbumin
a-, b-casein
a-lactalbumin b-
lactoglobulin
Detection of allergen-specific antibodies
in sera and saliva
Diamond-like carbon coating [41]
Fluo-larray – Development of nanoparticle-based
amplification approach for fluorescence
based detection of IgE
Znse Nanocrystals [42]
Fluo-larray – Development of a microchip dedicated to
allergy diagnosis with magnetic
nanoparticles as IgE capture
nanoplatform
Magnetic nanoparticles [43]
Fluo-larray – Development of model system for
detection of allergen-specific IgE
antibodies and for mast cell activation
profiling
Nanopatterned microchip [44]
CL-larray Egg white, cowÕs
milk, wheat,
buckwheat,
peanut, soybean
Automated multiplex detection of specific
IgE
None [45]
DPV – Development of aptamer-AuNPs
conjugates-based biosensor for
quantitative detection of IgE
Gold nanoparticles [53]
EIS and
amperometric
Ara h2 epitope Comparison of impedance and
amperometric strategies for allergy
diagnosis.
Gold nanoparticles [58]
QCM – Development of aptamer-based
piezoelectric biosensor for quantitative
detection of IgE in human serum
None [63]
FPW-MEMS – Development of a paired microsystem for
quantitative detection of IgE in human
serum allergy
None [64]
FPW-MEMS – Prototype for point of care allergy
diagnosis
None [65]
Abbreviations: SPR, Surface-plasmon resonance; LS, Light scattering; Fluo-larray, Fluorescence-based microarray; CL-larray, Chemilumines-
cence-based microarray; DPV, Differential pulse voltammetry; EIS, electrochemical-impedance spectroscopy; QCM, Quartz-crystal microbal-
ance; FPW-MEMS, Flexural plate-wave micro-electromechanical systems.
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A further example of immuno-magnetic nano-beads
used in an optical fiber SPR biosensor was reported by
Pollet et al. [22]. A nanobead-based sandwich assay was
evaluated for the fast, accurate detection of peanut
allergens. In comparison with other assay formats (see
Fig. 3a), the sensitivity enhancement by integration
MNPs was particularly significant giving an improve-
ment in the LODs in complex food matrixes of two orders
of magnitude.
The investigations presented so far were all focused on
simple allergen detection, but, following the general trend
of food technologies, increasing attention is being paid to
the development of innovative platforms with greater
automation and multiplexing capabilities than traditional
biological binding assays [23,24]. More and more prod-
ucts nowadays contain many processed ingredients,
which are often shipped from different parts of the world,
and share common production lines and storage spaces.
Multi-analyte detection has therefore become an urgent,
challenging task. SPR-biosensor technology, in multi-
channel or array-imaging instrumental set-ups, offers a
useful tool to high-throughput detection.
Simultaneous quantitative determination of five whey
proteins in six samples was performed by Billakanti et al.
[25] for various whey and milk sources. The ProteOn
XPR36 protein-interaction-array system (Bio-Rad Labo-
ratories, Hercules, CA, USA) was used to provide up to
36 interactions (6 ligands and 6 samples) to be moni-
tored simultaneously, with six horizontal and six vertical
channels. The biosensor performances were assessed in
terms of precision and accuracy, giving a relative stan-
dard deviation lower than 4%, and cross-reactivity and
non-specific binding were both found negligible.
Rebe Raz and co-workers [26] used i-SPR technology
for the rapid, quantitative detection of multiple food
allergens. An angle-scanning i-SPR system was com-
bined with an antibody microarray directed against 13
major food allergens (see Fig. 3b and c). A continuous-
flow microfluidic (CFM) spotter was employed to gener-
ate the microarray. The CFM spotter applied a micro-
fluidic interface to enable multi-step surface
functionalization on each spot and sequential deposition
of biomolecules in high-quality spots on hydrophilic
surfaces. Due to the ability of the spotter to address each
spot, which was kept isolated from its surroundings, the
CFM spotter offers many advantages over the conven-
tional spotting techniques in terms of minimized cross-
talk and background signals. After a preliminary bio-
sensor-performance evaluation with allergen-protein
extracts (see Table 1 for details), complete allergenic
profiles of commercial cookies and dark-chocolate prod-
ucts from different manufacturers were achieved. The
allergen profiles obtained with direct measurements re-
vealed different fingerprints for each food sample, thus
providing valuable information for both manufacturers
and monitoring authorities on the potential allergenicity
of the food products.
Several further investigations, not specifically aimed at
allergen detection deserve to be mentioned. Indeed, born
as molecular interaction monitoring technique, SPR
technology was successfully exploited for structural
characterization, providing useful information funda-
mental for allergen-risk management. For example, the
topography of the casein-micelle surface was investi-
gated by following the interaction between several
monoclonal antibodies specific for different epitopes of
casein (a
s1
,a
s2
,b, and k) [27]. IgE-binding activity of
peanut (Arachis hypogaea) lectin and legume lectins was
investigated by analyzing the sera from peanut-allergic
patients, and the correlation between cross-reactivity
and occurrence of structurally-related epitopes was fi-
nally assessed [28]. High-affinity aptamers were selected
by SPR characterization, specifically recognizing food-
allergen Lupin an 1 (b-conglutin) [29] and human-
immunoglobulin E (IgE) [30]. IgE is a trace component of
human serum closely involved in mediating allergic
reactions. In allergic patients, total IgE levels can be
correlated to the severity of the allergic disease, so,
detecting and quantifying IgE present in human sera
represents an efficient approach to allergy diagnosis.
Several SPR-based biosensors were developed with this
aim, some being proof-of-concept investigations for the
nanomaterial-based signal amplification by means of
gold localized plasmon-resonance coupling [31–33] or
light scattering [34,35] (see Table 2).
2.2. Other optical detection
Although SPR is considered the main tool to monitor
biomolecular interactions, for high-throughput multi-
plexed analysis, many studies still rely on array-based
methods, employing fluorescence or colorimetric detec-
tion.
Chen et al. [36] developed a highly efficient assay to
identify eight food allergens simultaneously on the surface
of optical thin-film biosensor chips (i.e. soybean, wheat,
peanut, cashew, shrimp, fish, beef, and chicken). Specific
allergen DNA was isolated and amplified by two tetraplex
PCR systems that were developed and validated in the
same study, followed by an enzyme-labeled indirect
detection. The enzymatic reaction products precipitated
on the thin-film surface and modified the interference
pattern of light on the biosensor surface, producing a
significant color change on the surface. The main
advantage of this approach was the possibility to perceive
the color change with the naked eye, thus avoiding re-
course to sophisticated equipment and related costs. An
example of the multiplexed allergen detection achievable
in commercial food products with this optical chip is
shown in Fig. 4. Similar results on a different combination
of allergens (i.e. mustard, lupine, walnut, hazelnut, celery,
Trends in Analytical Chemistry, Vol. 47, No. , 2013 Trends
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almond, oat, and sesame) were reported by the same
research group a few months later, further assessing
biosensor applicability (see Table 1 for further details)
[37].
Another DNA-based microarray method was devel-
oped by Maquieira and co-workers [38]. An example of
integrating PCR amplification with digital versatile disk
(DVD) technology for multiple-allergen detection was
presented [38]. Three relevant allergens (i.e. hazelnut,
peanut, and soybean) were chosen, since they were
considered to be the most difficult allergens to analyze,
especially by protein-based techniques, due to extraction
issues. Appropriate positive and negative controls were
implemented in the array to guarantee high-quality re-
sults. Indirect enzyme-labeled detection was accom-
plished, exploiting the modification of the laser-signal
intensity reaching the detector by enzymatic precipita-
tion, correlated with the amount of amplified sequence.
Optical properties of engineered NPs provided another
labeling tool for the development of indirect detection. A
heterogeneous immunoassay for the determination of soy
protein was reported for the first time using Nile Blue (NB)-
doped silica NPs. Following previous results obtained with
antibody functionalized gold NPs (AuNPs) in an homo-
geneous immunoassay, the method presented by Go
´mez-
Hens and co-workers in 2011 was a long-wavelength
fluorimetric immunoassay using a conjugate composed of
anti-soy protein antibodies bound to NB-doped silica NPs
[39]
.
The immunoassay was developed in 96-well
microplates using a competitive format with antibody
capture. Soy proteins were immobilized onto the wells and
bovine serum albumin was added to block the surface,
thus minimizing non-specific binding. The immunoassay
was applied to the analysis of food containing soy protein,
providing reliable results not statistically differing from
results obtained with a commercial ELISA kit.
Figure 4. (A) An optical thin-film biosensor with capture probes spotted by a computer-controlled dispenser. (B) Results of commercial food
products analyzed by the biosensor-chip method: 1. Cereal bar (cashew, wheat, and beef); 2. Chocolate chips (wheat, soy, and beef); 3. Wheat
biscuit (wheat and beef); 4. Dark chocolate (soy, chicken, and beef); 5. Fried mud carps with fermented soybean (wheat, soy, and fish); 6-blank
control (ddH
2
O) {Reprinted with permission from [36],ªCopyright 2011 American Chemical Society}.
Trends Trends in Analytical Chemistry, Vol. 47, No. , 2013
20 http://www.elsevier.com/locate/trac
Complementary to the detection of allergens, several
optical microarrays have been devised to give more
insights into epitope characterization and allergy diag-
nosis [40–45]. Some of the most recent achievements in
these fields are summarized in Table 2.
3. Electrochemical biosensors
By definition, an electrochemical biosensor is a self-
contained integrated device, which is capable of provid-
ing specific quantitative or semi-quantitative analytical
information using a biological-recognition element,
which is retained in direct spatial contact with an elec-
trochemical transduction element [46]. Due to the low
cost and ease of miniaturization, electrochemical bio-
sensors hold great promise for particular applications
where minimizing size and cost is crucial [e.g., on-line
allergen-contamination monitoring and point-of-care
(POC) allergy diagnosis].
Electrochemical biosensors can be classified into
potentiometric, amperometric, voltammetric and
impedance types. Potentiometric sensors have been tra-
ditionally defined as a zero-current devices that measure
the potential across an interface, often a membrane. By
contrast, both amperometry and voltammetry are based
on the measurement of current as a function of applied
electrode-solution voltage. During amperometric mea-
surement, the working electrode (i.e. the sensing elec-
trode) is held at constant potential, while the current is
monitored as a function of time and related to the con-
centration of the analyte. During voltammetric mea-
surement, the current of the working electrode is
recorded as a function of the potential. Several types of
experiment may be performed to gather information
from voltammetry (e.g., cyclic, linear sweep, square
wave, stripping and pulse).
Impedance biosensors measure the electrical imped-
ance of an interface by imposing a small sinusoidal
voltage at a particular frequency and measuring the
resulting alternating current. The current-voltage ratio
gives the impedance. The impedance of the interface
may be measured at a single frequency or at different
frequencies, the latter approach known as electrochem-
ical impedance spectroscopy (EIS). Biosensors can be
designed with Faradaic or non-Faradaic approaches. In
Faradaic-EIS, a redox probe is alternately oxidized and
reduced by the transfer of an electron to and from the
metal electrode. The specific analyte binding is detected
as change of the charge-transfer resistance. By contrast,
non-Faradaic-EIS employs no redox probes, and imped-
ance changes derive primarily from displacement of
water and ions by target molecules binding to the sensor
surface. Given that no additional reagent is required,
non-Faradaic schemes are somewhat more amenable to
POC applications.
The first applications of electrochemical biosensors to
food-allergen management date back to 2008 [47,48].
In the following sub-sections, the most relevant investi-
gations have been selected and classified according to the
transduction mode employed.
3.1. Potentiometry, amperometry and voltammetry-
based detection
Starting from the preliminary results achieved by elec-
trochemical platforms for allergen detection, chrono-
amperometry and voltammetry-based biosensors were
developed with different degrees of success. Micromate-
rials and nanomaterials were implemented as electrode-
surface modifiers or allergen labels, so as to improve the
sensing performance.
Direct allergen detection was described by Cao et al.
[49] by means of differential pulse voltammetry (DPV)
onto a nanocomposite-modified electrode. The stepwise
representation of the electrode modification is shown in
Fig. 5a – poly(L-arginine)/multi-walled carbon nanotube
(P-L-Arg/MWCNT) composite film was used to modify
the glassy-carbon electrode (GCE) through electropoly-
merization of L-arginine. Subsequently, AuNPs were
adsorbed on the modified electrode in order to immobilize
the casein antibody and to construct the final immuno-
sensor. The assembly process was systematically char-
acterized and, under optimized conditions, peak currents
of the redox couple decreased linearly with increasing
casein concentrations due to the formation of antibody–
antigen complex on the modified electrode. The advan-
tage of the proposed immunosensor relied on the ease of
fabrication of the nanocomposite electrode, which ben-
efited from the increased conductibility and amount of
antibody immobilization, providing good detection
reproducibility.
Similarly, DPV was employed for the detection of
another milk allergen, b-lactoglobulin, by means of
graphene-modified screen-printed electrodes. The interest
here focused on the graphene functionalization, particu-
larly the ability, through aryl diazonium salt electro-
grafting, to attach an organic film to the graphene surface
in a controlled manner. The immunosensor obtained
using this novel approach enabled an LOD with standard
buffer solutions of 0.85 pg/mL and a dynamic range of
0.001–100 ng/mL. Noteworthily, the immunosensor
was also tested in different matrices, including cake,
cheese snacks and sweet biscuits, after extraction with
standard buffers, and showed good correlation with the
results obtained from commercial ELISA kit [50].
Indirect allergen detection has been performed using
enzyme or NP labeling.
Marrazza and co-workers [51] reported the develop-
ment of an enzyme-linked chronoamperometric geno-
sensor using a commercially-available microfluidic-
based platform. The recognition of target DNA was
performed on paramagnetic micro-beads via sandwich
Trends in Analytical Chemistry, Vol. 47, No. , 2013 Trends
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hybridization with a capture probe (immobilized onto the
micro-beads) and a biotinylated signaling probe (see
Fig. 5b). After enzyme labeling of the biotinylated hybrid,
the beads were introduced in a disposable multichannel
cartridge and magnetically trapped. The quantitative
detection of the allergen was achieved based on moni-
toring the enzymatic kinetics. Some details about the
sensing performance are summarized in Table 3. The
advantage of the proposed configuration relied on the
possibility of measuring DNA sequences at the nM level
with high reproducibility and potential multiplexing
capability.
NP labeling is based on the dissolution in acidic
medium of the NPs, after the specific bio-recognition,
followed by the detection of the metal ions by sensitive
electroanalytical techniques. Following this approach,
Xu and co-workers [52] developed an electrochemical
biosensor for the detection of chicken ovomucoid
(CHOM) based on ZnO quantum dots (QDs) (see Fig. 5c).
The ZnO-QD/CHOM self-assembled bio-conjugate was
captured on a concavalin A-modified electrode. After
acidic dissolution, the electrode was subjected to square-
wave voltammetry and the zinc stripping peaks moni-
tored as a function of the CHOM concentration.
Figure 5. (A) The multilayer structured immunosensor fabrication process. (B) The bead-based assay. (C) Assembly and electrochemical strategy
for detection of chicken ovomucoid. (Abbreviations: MWCNTs, Multi-walled carbon nanotubes; Arg, Arginine; AuNPs, Gold nanoparticles; Ab,
Antibody; BSA, Bovine serum albumin; QDs, Quantum dots; CHOM, Chicken ovomucoid; Con A, Concavalin A).
Trends Trends in Analytical Chemistry, Vol. 47, No. , 2013
22 http://www.elsevier.com/locate/trac
Table 3. Electrochemical and electromechanical biosensors for detection of food allergens
Transduction Analyte Receptor Assay Matrixes Nanomaterial LOD Ref.
DPV (direct detection in
presence of redox probe)
Casein Polyclonal Ab Direct Cheese AuNPs + Poly(
LL
-
Arg)/ MWCNTs
50 ng/mL in
standard solutions
[49]
DPV (direct detection in
presence of redox probe)
b-lactoglobulin Ab Direct Cake, cheese snacks, sweet
biscuits
Graphene 0.85 pg/mL in
standard solutions
[53]
DPV (indirect detection,
alkaline phosphatase label)
Hazelnut genes:
Cor a 1.03,
Cor a 1.04
DNA Sandwich Hazelnut, Chocolate, Soy
milk, Chocolate cream,
Biscuit, Breakfast cereal,
Lecithin supplement,
Ketchup, Peanut butter,
Snack, Peanut
None Cor a 1.03: 0.3 nM
Cor a 1.04: 0.1 nM
[47]
RT-CA (indirect detection,
alkaline phosphatase label)
hazelnut gene:
Cor a 1.04
DNA Sandwich – Magnetic
microbeads
0.2 nM [51]
SWV (indirect detection,
dissolution of ZnO-QDs)
Chicken ovomucoid Con A Direct – ZnO QDs (3 nm) 1 ng/mL [52]
f-EIS (direct detection in
presence of redox probe)
Peanut Ara h1 Monoclonal Ab Direct – None 0.3 nM [48]
f-EIS (direct detection in
presence of redox probe)
Peanut Ara h1 Monoclonal Ab Direct – None 0.08 nM [54]
f-EIS (direct detection in
presence of redox probe)
Peanut Ara h1 DNA Indirect Peanut milk beverage None 0.35 fM in standard
solutions
[57]
Faradaic single frequency
impedance measurement
(direct detection in presence
of redox probe)
Peanut protein Ara h1 Polyclonal Ab Direct Canned soup None NA [55]
nf-EIS (direct detection) Peanut protein Ara h1 Polyclonal Ab Direct – 3 nm Au coated
nanoporous
membrane
NA [56]
QCM Shrimp Polyclonal Ab Direct spiked water samples AuNPs (10 nm) 0.333 lg/mL [61]
QCM Gliadin Polyclonal Ab Direct Wheat, barley, oat, rice,
foxtail millet, corn,
buckwheat, soybean,
pancake mix, custard mix,
baby rice, buckwheat, rye
biscuit, digestive biscuit,
plain crackers, red date
wheat crispy biscuit, cream
cracker, crisp flakes of rice
and wheat
AuNPs (25 nm) standard solutions:
8 ppb
food matrix:
1 ppm
[62]
Abbreviations: DPV, Differential pulse voltammetry; RT-CA, Real-time chronoamperometry; SWV, Square-wave voltammetry; f-EIS or nf-EIS, Faradaic or non-Faradaic electrochemical-
impedance spectroscopy; QCM, Quartz-crystal microbalance.
Trends in Analytical Chemistry, Vol. 47, No. , 2013 Trends
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Finally, a DPV-based biosensor was developed for the
ultrasensitive detection of IgE in human serum (see
Table 2)[53]. Aptamer-AuNP conjugates were imple-
mented as the sandwich-amplification element and
accumulation reagent of Methylene Blue, used as the
electrochemical indicator. Good linear correlation was
obtained for the detection of human IgE over the range
1–10,000 ng/mL with an LOD as low as 0.52 ng/mL.
An important disadvantage shared by all the above
electrochemical biosensors was off-line nature of the
measurements. The sensing area was usually incubated
with the analyte solution for times ranging from 30 min
to 2 h, rinsed and then employed for the electrochemical
measurement, so it was not real-time detection.
3.2. Impedance-based detection
Impedance biosensors are often considered as the class of
electric biosensors that show great promise for POC
applications due to their low cost, ease of miniaturization
and label-free operation. Suni and co-workers in 2008
developed the first impedance biosensor for food-allergen
detection (see Table 3)[48]. It represented a proof-of-
concept study for the detection of peanut-protein Ara h1
in standard solutions, providing a LOD lower than
0.3 nM. Initially demonstrated on a gold electrode, the
same approach was confirmed in a second investigation
by replacing gold with degenerate Si as electrode, with a
marked improvement in performance [54]. Several
advantages were expected for degenerate-Si electrodes
compared to other electrode materials (e.g., the more
stable linking chemistry than thiol-gold chemistry
proved to oxidize rapidly in ambient conditions). More-
over, Si electrodes can self-passivate, since exposed sur-
face silicon sites (i.e. sites not involved in Si–C bonds)
rapidly oxidize and become electrochemically inert.
Afterwards, a step towards food-matrix detection was
carried out by the same group considering methods to
reduce or to compensate for non-specific adsorption
during the detection of peanut-protein Ara h1 in canned
soup. Sample dilution and blocking of un-reacted surface
sites on a gold electrode with bovine serum albumin
were assessed to reduce non-specific adsorption dra-
matically [55].
In another work, Suni and co-workers reported the
development of a nanopore immunosensor by covalent
immobilization of peanut antibody within gold thin-film-
coated pores of commercial polycarbonate membranes
[56]. Protein Ara h1 was detected in terms of conduc-
tivity change depending on the partial pore occlusion by
antigen binding. The influence of different pore sizes was
evaluated but no unequivocal conclusions on optimal
geometry were drawn.
Electrochemical DNA-based biosensors have been
widely used for detecting specific genes. In order to cope
with the crucial step of DNA-probe immobilization, sur-
face-confined stemloop DNA structures have been
designed and proposed as capture probes with perfor-
mance superior to linear probes in terms of ability to
discriminate mismatches. Basing on this approach a
DNA sensor for detecting peanut-allergen Ara h 1 was
developed by Sun et al. with a dually-labeled probe (i.e. a
thiol group at its 50end and a biotin tag at its 30end).
The stem-loop probe was ‘‘closed’’ when the target oli-
gonucleotide was absent, and ‘‘open’’ after hybridization
with the target, thus moving away from the electrode
surface the biotin group at its 30end. The electron-
transfer efficiency changes resulting from the detach-
ment of biotin tags from the electrode surface were
proved by Faradaic EIS. The LOD of this method was
0.35 fM with the linear response in the range 10
15
–
10
10
M with the ability to discriminate a mismatch of
one base. The proposed strategy has been successfully
applied to detect Ara h 1 in DNA extracts of peanut-milk
beverage [57].
An interesting investigation carried out by Rusling
and co-workers was aimed at developing a peptide-based
bioelectronic sensor array to measure multiple anti-
peanut IgE accurately [58]. The sensors featured a syn-
thetic peptide layer of the major IgE-binding epitope from
Ara h2 attached to a dense AuNP film on a pyrolytic
graphite electrode. Faradaic and non-Faradaic imped-
ance and amperometric detection strategies were com-
pared, with the aid of labeled secondary antibodies,
when required (see Table 2). Non-Faradaic-EIS with
biocatalytic amplification gave the best analytical figures
of merit along with an excellent LOD of 5 pg/mL for IgY
in serum.
4. Electromechanical biosensors
Quartz-crystal microbalances (QCMs) and micro-elec-
tromechanical systems (MEMS) have emerged in recent
years as versatile biosensors, demonstrating remarkable
achievements (e.g., high sensitivity and label-free
detection) [59,60]. However, only a few examples of the
application of such approaches to allergen management
are available.
In QCM biosensors, the biological-recognition event
generates a mass change of the sensing layer, giving rise
to a change in the resonant frequency of the microbal-
ance. The application commonly requires the immobili-
zation of antibodies or DNA fragments (specific
complementary sequences or aptamers) on the trans-
ducer surface. In addition, AuNPs can be integrated both
as labels in sandwich assays (in order to enhance the
surface stress and the mass change of the immuno-
complex) and as surface modifier onto the quartz crystal
(in order to increase the amount of immobilized bio-
molecules). Focusing on allergen detection, the latter
approach was preferred – the large surface-to-volume
ratio of AuNPs was exploited to generate high-density
Trends Trends in Analytical Chemistry, Vol. 47, No. , 2013
24 http://www.elsevier.com/locate/trac
recognition layers and three-dimensional orientated
receptors, thus achieving an higher probability of
occurrence for specific interactions.
In particular, nanogold-modified film, developed by
Xiulan and co-workers by means of 1,6-hexanedithiol
and AuNP self-assembly, was aimed at real-time QCM
detection of shrimp allergen in food [61]. The sensor-
chip-surface modification was optimized by tuning sev-
eral parameters. In a direct immunoassay approach, an
LOD of 0.333 lg/mL for purified allergen solutions was
accomplished, with a run time less than 10 min.
Similarly, a QCM biosensor incorporating AuNPs was
developed by Wen and co-workers for the detection of
gliadin [62]. The frequency changes and the assay sen-
sitivity were compared with those of a conventional
QCM with a gold electrode unmodified. Experimental
results revealed that the integration of AuNPs increased
the overall sensitivity in standard solutions (LOD down
to 8 ppb). Also, the AuNP-modified QCM system was
applied to the detection of gliadin in several real samples
providing, in combination with a proper extraction
procedure, an LOD of 1 ppm, which is lower than the
official limit of gluten-free foods set by the European
Commission.
Further electromechanical biosensors discussed in the
literature aimed at allergy diagnosis. Proof of concept of
an aptamer-based piezoelectric QCM-biosensor array was
reported by Yao et al. for quantitative detection of IgE in
human serum [63]. Wang and co-workers designed and
characterized a flexural plate wave-biosensing micro-
system, optimized for the quantitative detection of IgE in
human serum [64,65].
5. Conclusions
In this article, we reviewed the most recent literature on
biosensors development for food allergen management
using a transduction-based classification.
Boasting high surface sensitivity and real-time re-
sponse, optical detection remains the preferred tech-
nique for quantitative diagnosis, also from the
perspective of easier multiplexing, since a commercial
CCD camera and other image sensors could detect
hundreds of reactions simultaneously. Combining the
selectivity of biology with the processing power of
modern microelectronics and optoelectronics offers
powerful analytical tools to be applied to the food-
safety field. Most of the optical biosensors are based
upon measurement of changes in the surface proper-
ties of a sensor-chip device, thus allowing detection of
analytes in complex matrices with minimal sample
pre-treatment. Despite the ease of multiplexing, mini-
aturization of conventional optical equipment into a
portable, robust system is not always straightforward
and, above all, cost effective.
In perspective, electrochemical biosensors boast the
promise of having advantages over optical devices in
providing low-cost, small-sized and disposable sensing
chips. However, the results achieved so far are still quite
far from this goal.
Off-line measurements, long incubation times and
sensitivity to non-specific binding from matrix compo-
nents are common open issues, which often constrain
the use of electrochemical biosensors to preliminary
proof-of-concept investigations on standard solutions. A
great deal of effort should be devoted in this direction in
order to exploit the features and the potential of elec-
trochemical biosensors in screening for food allergens.
Analogously, nanomaterial-based biosensors were
shown to be promising tools for improving sensing per-
formance, and are still in their infancy in their applica-
tion to food matrixes. Detailed investigations on the
interferences in real sample analysis and evaluation of
technological issues related to the final application to
food must be addressed before biosensors can fully ben-
efit from integrating nanotechnology.
Acknowledgements
This work was supported by a dedicated grant from the
Italian Ministry of Economy and Finance to the National
Research Council for the project ‘‘Innovazione e sviluppo
del Mezzogiorno - Conoscenze Integrate per SostenibilitaÕ
ed Innovazione del Made in Italy Agroalimentare -Legge
n. 191/2009’’.
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