Microchim Acta (2009) 166:1-19

DOI 10.1007/s00604-009-0165-z

REVIEW ARTICLE

Analytical nanotechnology for food analysis

Mayra Granda Valds
Arstides Camilo Valds Gonzlez
Josefa Angela Garca Calzn Marta Elena Daz-Garca

Received: 9 March 2009 /Accepted: 15 April 2009 /Published online: 12 May 2009
Springer-Verlag 2009

Abstract Nanotechnology involves the study and use of

materials at nanoscale dimensions (nanomaterial sizes of
<100 nm), exploiting the different physiochemical properties
exhibited by these nanomaterials from the same materials at a
larger scale. Nanotechnology is being demonstrated to have a
large impact on many aspects of food and agricultura!
systems, from the development of new food packing materials
to nano-delivery systems, including the analytical control of
the whole food chain. In fact, the need to generate fast, reliable
and precise information on the quality and security of
foodstuffs and food industry has resulted in an intensive
search for more selective and sensitive analytical methods.
Nanotechnology is one way to achieve these goals. Although
analytical nanotechnology applied to food industry is still an
emerging field, chemical sensor and biosensor technology for
use in this area has rather early taken advantage of the unique
merits of nanotechnology and nanomaterials. This article
reviews the recent progress made in analytical nanotechnology as applied to the food industry and to food analysis, with
particular emphasis on nano-sensing. A brief description of
the various nano-based sensing approaches is given and their
capabilities and limitations are discussed. Typical examples
are presented for exogenous compounds (e.g. pesticides, toxic

M. G. Valds
Department of Analytical Chemistry, University of La Habana,
La Habana, Cuba
A. C. Valds Gonzlez
Institute of Materials Science and Technology,
University of La Habana,
La Habana, Cuba
J. A. Garca Calzn M. E. Daz-Garca (El)
Department of Physical and Analytical Chemistry,
University of Oviedo,
Av. Julin Clavera, 8,
ES-33006 Oviedo, Spain
e-mail: medg@uniovi.es

anions, ripening gases or vitamin supplements) and endogenous compounds (from microorganisms to vitamins) in food.
In addition, selected nanotechnology-based analytical methods other than sensing are described.
Keywords Nanotechnology Food Analytical chemistry
Nano-based sensors

Introduction
The advent of Nanotechnology, introducing control over
matter at the nanometer scale, has produced a new class of
materials with novel properties, creating new possibilities in a
diversity of domains. Although there is currently a fierce
debate on the potential applications of nanotechnology in the
food industry, it is already taking advantage from the
versatility of nanoscience (Fig. 1). Research involving
nanotechnology to improve conventional performances and
to create unprecedent functions of foods are flourishing and
numerous examples have been developed in recent years.
For the food industry, nanotechnology applications include
among others: functional foods that contain nutrient-rich
nanomaterials or that reduce absorption of cholesterol,
intelligent packaging materials that release a preservative when
food is beginning to spoil, food packages that contain nanoparticles effective in destroying microorganisms, packages that
use biodegradable and environment-friendly material, plastic
films that provide barrier protection by preventing gases such
as oxygen and ethylene from damaging the food content,
fridges with nano silver coatings that keep out bacteria and
fungus, nanoscale particles that can bind to harmful matter and
remove potentially harmful compounds in food, devices
enclosing nanoceramic catalytic inserts which prevent cooking
oil from clumping, nanoparticles in chicken feed that latch on
bacteria and are then excreted by the birds so they are safer to
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4Analytical
2
nanotechnology for food analysis

M.G. Valds et al.

3

Nanotechnology in food industry

">>
Agriculture:
Product:-

Soil remediation
Water purification

Nano-additives

Jur

Nanosensors
biosecurity:
Nanosensor
s

Nanomaterials:

Nanosprays
Supplements

Conservation/Packagin
g

Fig. 1 Potential applications of Nanotechnology in the food industry

eat, encapsulation of nutrients in nanoscale spheres so they

enhance the biological activity of dietary supplements, nanomaterials added to PVC films that can prevent spoilage by UV
light, new containers infused with antibacterial silver nanoparticles that maintain food fresh and extend shelf life. Proof
of all this is the fact that the market for nanotechnology food
and food processing is currently valued at over two billion
dollars and is projected to grow to over 20 billion by 2010 [1].
Analytical chemistry plays an important role in the food
industry, both in the control of its quality as in its safety. Yet
relatively few are the nano-based analytical methods that have
been developed to control analytes of interest in foods. The
analytical aspect of the nanotechnology applied food industry
is just starting to emerge. The main branch of analytical
chemistry that has so far taken advantage of the unique merits
of the nano scale is that of sensors and, particularly, biosensors
(Fig. 2). Notwithstanding, at present few are the devices
devoted to food applications available commercially, most of
which are produced by the nanoelectromechanical systems
technology and are devoted to control the storage environment acting as active "sell by" devices.

However, according to Luong et al [7], it is still uncertain if

the increased capability of nanosensors is sufficient to open up
large markets. All there papers are of mandatory reading for
any analytical chemist interested in the subject.
In this article a brief overview on recent progress in
analytical nanotechnology applied to the food industry is
described, with particular emphasis on nano-sensing while
other analytical methods, not strictly included into sensing
but based on nanomaterials, will also be briefly mentioned.
To date there are several types of food analytes that are
being determined using nanosensors, among which are the
following: pathogens and mycotoxins, chemicals produced
by foods, food contaminants and compounds that measure
food quality. The first are by far the most investigated and
therefore this review will have two main parts: the first
dedicated to pathogens and mycotoxin analysis and a second
for the remaining analytes. Some of the described nanosensors are under research by several companies and, in
most cases, scientific details are not disclosed by manufacturers. Therefore, we shall refer to some of the more
accessible and more recent scientific information available
in each case. In the second part, some analytical applications
that do not refer to sensing will be mentioned briefly. It is
our hope that this review will be a useful information for
analytical chemists of different fields to start to focus on
nanotechnology in order to develop more reliable, faster and
simpler methods of analysis applied to the food industry.

Nano-sensing of pathogens and mycotoxins in

foods

Microorganisms are the worst source of contamination of

foods because they can produce changes that render them
Nano-based
analytical approaches

Sozer and Kokinei [2], in their review devoted to the

developments in the field of biosensors and bioelectrochemistry, stressed the fact that Nanotechnology is being used to
improve sensor performance and to develop biosensors based
on new detection techniques and that their use is spreading
into all amas of food industry. Huang and Choi [3] also
reviewed the use of nano structured materials in chemical
sensors and outlined their special features. The authors
considered that their performance characteristics (high sensitivity, fast response and recovery, potential for integration of
addressable arrays in massive scale, among others) set them
apart from other technologies present today. Other reviews [46] on the matter describe the nature of different nanostructures
and their use in the development of biosensors as analytical
tools, their evident advantages and limitations, as well as the
problems related to real-world applications of nanosensors.

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( Nan oi cle-based
sensors

Fig. 2 Analytical nanotechnology in the food sector

unfit for human consumption and can cause serious

infections or poisoning if consumed [8]. It is well known
that food and water contaminated with bacteria can
produce a number of foodborne and waterborne diseases
[9]. Conventional microbiological control of these microorganisms, although very reliable, is complicated. Microbiological methods rely mostly on the use of enrichment
and selective growth media to isolate and enumerate
viable bacterial cells from food samples, followed by
serological and biochemical confirmatory tests [10]. They
are also lengthy (long incubation periods of several days)
and when immediate clinical measurements have to be
taken, time is a very important element to consider if
people can become ill. On the other hand, failure to detect
pathogens in food can produce recalls which result costly
and reduce the confidence of consumers. Evidently, nano
scale devices that can detect toxins and pathogens in
foodstuffs (including beverages) in the lab or even better,
in the farm or during processing, in storage or on the shelf
represent, from the scientific point of view, a huge step in
sensing and from the commercial one, a huge step in the
food industry.
Arora et al [11] have reviewed developments in biomolecular electronic techniques for food pathogens.
Among them, special mention was given to nanomaterial based devices, but most of the papers referred
fundamentally to DNA detection. According to these
authors "sensors for pathogen detection in industrial food
process control...may be the market niches in which nanodevices will become established in the next few years...
Time is not far when these nano-devices would serve the
common peoples needs instantly." In a similar vein, A.
Baeumner in her review [12] pointed out that "research is
needed to develop nanomaterials that help integrate the
nano- and micro-world with the required macro-world of
food analysis....rapid, portable, highly sensitive, specific,
yet inexpensive detection technology will allow us to
overcome challenges we encounter today in pathogen
detection".

More recently, Lazcka et al [13] have reviewed methods

used for pathogen detection in the last 20 years, comparing
traditional methods with biosensing. According to these
authors, the food industry represents almost 40% of the
areas interested in pathogen detection. Salmonella, E. coli
and Listeria are among the microorganisms most studied in
the literature regarding detection and quantification as they
have been the cause of the largest outbreaks. When both the
estimated number of cases and mortality rate are considered
for bacterial, viral, and parasitic cases of foodborne illness,
Salmonella causes 31% of food related deaths, followed by
Listeria (28%), Campylobacter (5%), and E. coli 0157:H7
(3%) [14]. Unfortunately no nanomaterial-based sensors
were mentioned, but when mentioning new trends the

authors highlighted the advantages of a nano scale approach

in biosensing. Among them:
(a) the possibility of mass production and reduced unit
costs
(b) working with sample volumes in the range of nanoliters, thus using less reagents and making the cost of
the analysis not too high
(c) shorter analysis times
(d) multi-analyte analysis
(e) safer and environmentally friendly devices
E. coli 0157:H7
E. coli 0157:H7 was first discovered in 1982 and it is
estimated to cause over 70,000 cases of sickness and over
50 deaths a year only in the USA [15]. It produces toxins
that damage the lining of the intestine, causes anaemia and
stomach cramps, serious complications called haemolytic
uremic syndrome and hemorrhagic colitis [16], as well as
extra-intestinal infections that can spread throughout the
body. Human infections with E. coli 0157:H7 have been
traced back to food products that include meat, apple juice
or cider, milk, alfalfa sprouts, unpasteurized fruit juices,
dry-cured salami, lettuce, and cheese curds [17]. Its
infective dose is fewer than 100 organisms [18]; therefore,
schemes for its detection require some sort of amplification
or must be very sensitive per se. In this sense, several
approaches employing nano-sensing have been used to
unravel the problem. According with J. Wang [19], the
great interest in nanomaterials is due to the ability to tailor
the size and structure of these materials, and hence their
properties, offering thus excellent possibilities for the
design of novel sensing devices. Among these materials
are nanotubes, nanofilms and nanoparticles [19].

X. Mao et al [20] were the first authors to develop a

microgravimetric quartz crystal microbalance (QCM) DNA
sensor for the detection of E.coli based on nanoparticle
--
Array
biosenso
anoparticie
sin

(Electronic
nose

cantilever
s:

Microfluidi
c
device
/ Nano

Lab-on-a-chip

test-strips

amplification. The biotinylated target DNA was captured by

the single-stranded DNA (ssDNA) probe self-assembled on

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the transducer surface and the signal was amplified using

streptavidin-coated ferrofluid (Fe304) nanoparticles. Sensor
fabrication resulted a bit complicated, requiring self
assembly of a thiolated DNA probe unto the gold electrode
of quartz crystal. Blocking with BSA (bovine serum
albumin) was then carried out and afterwards the hybridization reaction with the biotinylated target DNA occurred.
In these conditions, the frequency change produced was
marginal but when the nanoparticles were injected, the
signal was significantly amplified. A quantitative relation
was found between signal and E. coli concentration. The
LOD reported (2,67x 102cfu mL-1) was lower than that of
previous QCM DNA based bacteria detection sensors, but
no real samples were analyzed. Authors concluded that the
use of the nanoparticles enhanced sensitivity due to their
relatively large mass (mass-enhancers) in comparison to the
DNA targets.
In general, nanotube-based sensors include carbon nanotubes (CNT) as the most popular, but also metal oxide- and
metal-tubes have been used. MerkoQi [21] and Fang et al
[22] have recently reviewed the state of the art of CNT in
analytical sciences and DNA electrochemical biosensors,
respectively, and considered that, because of their unique
mechanical, electrical and geometrical properties, they were
ideal as nanoscale sensors. Different studies [23, 24] have
shown that CNTs can enhance electrocatalytic activity and
reduce fouling on electrode surface, making them adequate
for electrochemical sensing. Their large specific surface
area can enlarge biosensor response.
Y. Cheng et al [25] used multi-walled carbon nanotubes
(MWCNT) to produce a Nafion-modified glassy carbon
electrode for the amperometric detection of coliforms,
represented by E. coli. The MWCNT were functionalized
with carboxylic acid groups and contacted with a Nafion
(p erfluorosulfonated cation-exchanger polymer) solution
containing the electrocatalyst. The modified glassy carbon
electrode was prepared by dropping a suspension of the
MWCNT/Nafion on the carbon electrode and allowing the
solvent to evaporate. The detection scheme was based on
the fact that galactosidase (an enzyme produced by E. coli
as part of its regular metabolic process and thus an indicator
of the presence coliforms), reacted with the substrate (paminophenol-(3-galactopyranoside) added to the bacterial
solution. The hydrolysis product, p-aminophenol, was
detected by the MWCNT/Nafion modified glassy carbon
electrode. After 10 min. of enzymatic reaction, the current
was recorded and the quantity related to the concentration
of E. coli in the sample solution. The effect of factors such
as pH, temperature and amount of MWCNT/Nafion
suspension on electrode surface were studied. The sensor
was reported to have a detection limit of 10 cfu mL -1 if
bacteria were incubated for 5 h. The method was used for
detection of E. coli in river water and authors recommended
its application in the food industry due to rapidity and
sensitivity.
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For the detection of E. coli, W. Zhang et al [26] also

employed a glassy carbon electrode modified with a
bismuth nano-film (BiNF) of 90 nm maximum thickness.
A flow injection (FIA) approach was used and the sensing
mechanism was similar to that reported by Y. Cheng et al
[25]. P-D-glucuronidase was released from the E. coli
strains when it was incubated in trytic soy broth containing
lauryl sulphate sodium and deoxycholate sodium. When the
substrate (4-nitrophenyl (3-D-glucuronidase) was added, the
enzyme catalysed its hydrolysis and 4-nitrophenol (4-NP)
was produced. Then, the liquid containing 4-NP was
injected into the FIA system and its reduction took place
at the BiNF electrode exhibiting a single well-defined
reduction peak at -0.53 V. The enhanced response current
was proportional to E. coli concentration and LOD of the
3-hour assay is reported as 100 cfu mL -1. The diminished
reduction potential and increased current signal when the
BiNF electrode was used gave place to enhanced sensitivity.
Seven different E. coli samples were assayed and interferente studies (for Salmonella, Staphylococcus aureus, among
others) were made. These authors recommended the potentialities of the method for rapid detection of E. coli not only
in the food industry but also in public health, clinical
diagnosis and environmental monitoring.
CNTs are not the only type of nanotubes used for
sensing of E. coli. Chan Cho [27] and co-workers have
fabricated an electrochemical immunosensor with selfassembled peptide nanotubes (PNTs) for the detection of
E. coli 0157:H7. First, cyclic peptides were used for the
self-assembly of the nanotubes. The resulting solution was
then deposited on the surface of the electrode (a graphite
paste printed on slide glass). The resulting electrode was
then put in contact with antibody solution (FITC-labelled
anti-E. coli) for 24 h, after which it was rinsed and dried.
The attachment of antibodies to PNTs reduced the
necessary steps for immobilization and, therefore, time
analysis. Cyclic voltammetry using a [Fe(CN) 6]344- redox
probe was used for sensing the antigen/antibody interaction
on the electrode surface. The features of the interfacial
properties of the immunosensor changed during this
interaction and the voltammetric behaviour of the probe,
thus detecting the presence of the pathogen. Although the
authors stressed as advantages of the sensor its instrumental
portability allowing field sampling, no real samples were
afforded.

For the rapid detection of E coli 0157:H7 in ground beef

an impedance biosensor based on an interdigitated array gold
microelectrode coupled with magnetic nanoparticle-antibody
conjugates was developed by Varshney and Li [28]. The
conjugates were prepared by immobilizing biotin-labeled
polyclonal goat anti E. coli antibodies onto streptavidincoated magnetic nanoparticles (Fe304/magnetite/Fe). E coli
0157:H7 present in the samples (previously inoculated with
diluted E. coli cultures) was bound by these labelled

nanoparticles. With the help of a magnetic field, the

nanoparticles were then concentrated onto the active layer
of an array microelectrode. No redox probe, chemical
immobilization or sample incubation was needed. In spite
of the fact that authors claimed that the use of the magnetic
field and the nanoparticles enhanced sensitivity due to the
formation of clusters between conjugates and cells, the LOD
was rather high (8 x 105 cfumL-1) in comparison with those
reported in the previously mentioned papers. However, real
food samples were analyzed using this nano-based biosensor.

Analytical
nanotechnology for food analysis
6

In a next step, these authors embedded the labelled magnetic

nanoparticles in a especially designed microfluidic flow cell
for impedance measurements. A lower LOD could be
obtained for E. coli determination in the same type of
sample only if a larger volume of sample was used [29].
E. coli is apparently the most food- and water-pathogen
investigated; however, other equally dangerous pathogens
(Table 1) need to be nano-sensed in foods. Unfortunately,
only a few papers could be found in the literature. We will
mention some of them.
Salmonella infantis
Salmonella Infantis, associated with the egg and especially
the chicken meat industry, has been of significant public
health concern in many countries, and remains so in some
regions. Salmonellosis is one of the most important
bacterial enteric diseases causing millions of human and
animal illnesses and significant economic losses worldwide. Although the most prevalent serotypes are S.
enteritidis and S. typhimurium, which together cause
about 90% of reported cases, in recent years S. infantis
became the most prevalent one and was responsible for
about 5% of the illnesses [30]. The economic impact of
Salmonella infections bears heavily on the cost of medical
care and premature loss of life. On the other hand, being
salmonellosis a zoonotic disease, the economic loss it
causes to the industry is also significant. Consequently,
analysis of this pathogen is of key importance in the food
chain and the food industry.
A network of single walled carbon nanotubes has been
used by Villamizar et al [31] for the construction of a fieldeffect transistor (FET) biosensor for Salmonella Infantis.
The recognition process was based on antigen-antibody
interaction. The CNTs were functionalized with antiSalmonella antibodies and Tween-20 was used to avoid
non-specific binding of other bacteria. Authors reported
that the adsorption of S. Infantis was selective, appearing as
small bright dots along the CNT (SEM image) and
producing a significant decrease in the electric current at
negative gate voltages. The device could detect at least
100 cfu mL-1 of the pathogen in 1 hour and studies were
underway to apply it to Salmonella contaminated food.

Vibrio parahaemolyticus
Vibrio parahaemolyticus (Vparahaemolyticus) is a bacterium in the same family as those that cause cholera. It lives in
brackish saltwater and causes gastrointestinal illness in
humans. Food poisoning outbreaks associated with V
parahaemolyticus have been reported throughout the world,
especially occurring in areas where people often consume
raw and undercooked shellfish in their daily diet. An

M.G. Valds et al.5

occur each year in the United States [32].

G. Zhao and co-workers [33] proposed a disposable
amperometric enzyme immunosensor for the detection of V
parahaemolyticus based on a screen-printed electrode
coated with a composite of agarose doped gold nanoparticles. This membrane served for the immobilization of
horseradish peroxidase (HRP) labelled V parahaemolyticus
antibody. The nanoparticles acted as very small conduction
pathways that permitted efficient and direct electron
transfer as the distance between the active site and the
electrode was greatly reduced. After preparation of this
immunosensor, the assay was based on reduction of the
cathodic peak current when microliters of a V parahaemolyticus suspension was dropped on the sensor and
incubated for 30 min at 25C . V. parahaemolyticus formed
an immunocomplex (with HRP-anti- V parahaemolyticus)
on the surface of the sensor and detection was based on the
inhibition of the enzymatic activity over the oxidation
reaction of thionine by H202 (both added after incubation
and before measurement). Authors studied selectivity for
other common food pathogens, although no food samples
were analyzed.

Micotoxins
Mycotoxins are toxic chemical products very harmful when
present in food. They are secondary metabolites naturally
produced by fungal species that grow on agricultural
products, such as nuts, fruits and grains, among others,
both in the field, after harvest and during storage [34].
Their presence in beverages and foods is due to direct
contamination of plants or their products or by contamination of animal feeds [35]. Table 1 shows some of the
possible effects in humans.
Many are the methods employed to detect mycotoxins in
complex food matrixes, but ELISA and chromatographic
methods are the most used [36]. More recent trends include
biosensing [37] and relatively few reports include nanosensing in food.
In 2007, Prieto and co-workers [38] reviewed emerging
biotechnological tools for mycotoxin analysis and stated
that nanotechnology has recently been incorporated into
mycotoxin assay. D. Yao et al [39] were among the few that
went into this field. After a previous preliminary study [40],
they constructed an electrochemical biosensor for sterigmatocystin by using multi-walled carbon nanotubes modified
with an enzyme, aflatoxin-detoxifizyme. MWCNTs were
used for enzyme immobilization after they were deposited
on a gold electrode. Although LOD was improved, the
authors themselves expressed the need for further investigation of this sensor due to the effect of temperature, pH
and other factors on detection and applicability.

estimated 4500 cases of V parahaemolyticus infection

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Table 1 Characteristics of some food-borne bacteria

Bacteria

Effects

Salmonella
Bacillus cereus

Salmonellosis, gastroenteritis
Diarrhea, emesis and non-gastrointestinal
diseases

Vibrio
parahaemolyticus
Listeria
monocytogenes
Campylobacter
jejuni
Mycotoxins

Diarrhea
Abortion or still deaths, flue
Diarrhea
Carcinogenic, immunosuppressive,
nephrotoxic, neurotoxic

Meat, poultry, non-pasteurized milk, dairy products

Milk products, raw vegetable sprouts, custards,
soups, cooked meats
Raw or undercooked shellfish (especially oysters), seafood
Ready to eat food, canned food, fruits, vegetables
Poultry, non pasteurized milk

Optical sensors based on

nano materials have been
much less applied to the
detection of analytes of
interest in the food industry.
Quantum dots (QD) are
practically the only nano
material used with this aim.
QD are nanocrystals of
inorganic semiconductors that
are somewhat restricted to a
spherical shape of around 2
to 8 nm diameter [41]. Their
fluorescent properties are
size-dependent and therefore
they can be tuned to emit at
desired
wavelengths
(between 400 and 2000 nm)
if synthesized in different
composition and size. In this
way, QDs of different sizes
can be excited with a single
wavelenght and emission
controlled
at
different
wavelenghts, thus providing
for simultaneous detection.
This, together with their
highly
robust
emission
properties, make them more
advantageous for labelling
and optical detection than
conventional organic dyes
[42]. Their high quantum
yields and their narrow
emission bands produce
sharper colours, lead to
higher sensitivity and the
possibility of multiplexing of
analysis [43, 44]. Costa et al
[45] have reviewed the
progress in exploiting these
novel probes in optical
sensing, as well as their still
unexploited
sensing
capabilities. In analytical
chemistry field their major
application has been as

11

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fluorescent labels while

applications to food analysis
is, up to now, unexploited.

Goldman et al [46, 47]

have
used
QDs
for
fluoroimmunoassays
of
toxins. They detected four
toxins simultaneously, three
of which are naturally
responsible for food or
waterborne sickness. The
CdSe-ZnS core-shell QDs
were
capped
with
dihydrolipoic
acid
and
bioconjugates
with
the
appropriate antibodies were
prepared. A sandwich immunoassay was performed in
microtiter plates where the
toxins and different QDs were
incubated for an hour.
Fluorescence was measured
at adequate wavelengths and,
although there was spectral
overlap, deconvolution of
spectra
revealed
fluorescence contribution of
all toxins. Signals increased
with toxin concentration in
different ranges according to
the particular toxin. No LODs
were reported. Although
authors
treated
the
bioconjugate
QDs
as
fluororeagents, they can be
considered as "chemosensing
devices".
Nano-based sensing
approaches for other
analytes of interest in
food

Some of the analytes of interest

for the food industry are food

Milk, eggs, plants and animal feeds

contaminants that can pass

through the food chain due to
their presence in soil, others
are components added to
foods to increase nutritional
values. There are also
compounds that are present
when food spoilage or
poisoning takes place or, on the
contrary, that must be present
when certain foods or
beverages are in optimum
state. Some of those
exogenous and endogenous
species whose concentration
must be controlled because
their presence modifies food
quality are described in the
following sections (Table 2).
Oxygen
Oxygen is one of the most
important
gases
from
environment because it may be
the reactant and also the product
in a major number of
chemical and biochemical
reactions.
Modified
atmosphere packaging (MAP)
is defmed as "the packaging
of a perishable product in an
atmosphere which has been
modified
so
that
its
composition is other than
that of air" [48]. In food

industry storage of foods

under vacuum or under
modified gaseous atmosphere
can maintain quality and
extend product shelf life by
slowing
chemical
and
biochemical
deteriorative
reactions. Examination of the
variation in residual oxygen
content of MAP or vacuum
packed foods in the period of
storage can be an important
indicator on the evolution of
degradative processes in food
products.
The
development of
nano-sensing methodologies
described for oxygen control
in packed foods is in its
early stage of development.
Gutierrez
et
al
[49]
developed a sensing device
that acted as an oxygen
indicator. A methylene blue
(MB) hybrid nanocomposite
material was deposited on
glass slides by a liquid
phase deposition technique
[50] obtaining a blue,
stable,
well
adhered,
optically
transparent
coating. When an upper
layer of triethanolamine
(TEA)
embedded
in
hydroxyethyl cellulose

Analytical nanotechnology for food analysis

Table 2 Nano sensing of some analytes of interest in foods

Analyte
Oxygen
Paraoxon

Source

Characteristics of nano-sensor

Allows aerobic microorganisms to

grow during food storage

Spin coating of TEA with an encapsulating polymer over

MB/TiO2 nano composite material
- Layer-by-layer films of chitosan, organophosphorous hydrolase
and thioglycolic acid-capped QD
- AChE encapsulated in the internal nano-environment of liposomes

Dichloros,
paraoxon
Parathion
Amitrole
Trimethylamine

Organophosphate pesticides found in soils

Ethylene
Xanthine and
hypoxanthine

Gas emitted in ripening process

Products formed during degradation of
meat or fish; used to evaluate freshness

Polyphenols

Present in wines and are of high nutritional

value due to anti-oxidant capacity
- In beverages used for sports practice
- In beer

Glucose

Components
of vinegars
Folic acid

Contaminant herbicide of soils and water

Gas produced during fish decay

Cyanide

Components that contribute to smell and

taste of Chinese vinegars
-For fortification of milk, fruit juices,
wheat flour
- Dietary supplements (tablets)
Biogenic polyamine that can cause
food poisoning
Accidental contamination of drinking water

Prion protein

Animal diseases (mad cow disease)

Spermidine

encapsulating polymer was

spin coated on the MB/TiO2
sensor under UV-irradiation,
the initially blue colour was
bleached. Authors explained
that photogenerated electron
holes were produced in the
semiconductor particles that
were able to irreversibly
oxidize the TEA, while the
electrons generated reduced
the MB to its leuco form. The
blue color began to appear as
soon as ambient oxygen reoxidised leuco-MB. Thus, a
colorimetric oxygen indicating
device was fabricated, but
authors point to a limited
stability of the device.
Although they suggested
the increased interest for the
development of oxygen
sensors in fields such as

Ref.
49
45, 52
54

- Zr02/Au nano-composite film electrodes

Carbon paste electrode modified with Fe(II)-phthalocyanine
- SnO2 nanoparticles doped with ZnO microrods
- Polyaniline-TiO2 nano composite

56
57
58
59

- Polyvinylpyrrolidone/ ZnO nanoparticles

Tungsten oxide-tin oxide nano composite
- Glassy carbon paste electrode modified with Au nanoparticles
and xanthine oxidase
- Nanocrystal gold-carbon paste electrode
Tyrosinase biosensor based on Au nanoparticle modified
glassy carbon electrodes
- GOx colloidal gold /CNT-Tefion composite electrode
- GOx entrapped in ionic liquid-DMF-AuNP composite
film-coated glassy carbon electrode
Electronic nose comprised of thick film gas sensors prepared
from ZnO nanoparticles doped with metal oxides or metals
-An ionic liquid-single walled CNT paste modified glassy
carbon electrode
-Single-wall carbon nano-tube film electrode
Sorption of double stranded DNA on polypyrrole nanofibers

60
62
63

Photochemically-generated AgNP on TiO2 coated on the

electrode surface of a PQC
Nanomechanical resonator array and secondary mass labelling

84

food packaging, no real

samples were analyzed.
In a recent paper,
Borisov and Klimant [51]
have developed luminescent
oxygen nanosensors based on
poly
(styrene-blockvinylpyrrolidone) nanobeads
stained with metal-ligand
complexes
whose
luminescence was quenched
by oxygen. The sensing
beads responded in real time
to
altering
oxygen
concentration
as
demonstrated
while
monitoring consumption of
oxygen during enzymatic
oxidation of glucose or while
monitoring dissolved oxygen
in a growing culture of E.
coli. The nanobeads were

64
65
68
71
89, 90
74
75
61

95

not applied to food analysis;

however, authors suggested
the applicability of the sensing
nanobeads for monitoring
oxygen
in
fermentation
media, in bioreactors and
biotechnology.
Pesticides
Pesticides
contribute
significantly
to
the
contamination
of
the
environment (soil, surface
and/or ground waters) due to
their extended use in many
areas of modem agricultural
production. Due to the high
water solubility of most
pesticides,
their
action
mechanism, their high level
of
toxicity
and
the

widespread
use
in
agriculture, there is a critical
need for highly sensitive and
selective analytical methods
for residue analysis of there
pollutants. Although there
are some examples of
nanosensors developed for
pesticides, scarce are the
papers
dealing
with
practical applications using
food samples. In the
following paragraphs the
recent research in this field
will be reviewed.
a) Paraoxon
Organophosphorous (OP)
compounds are a generation of
pesticides that are rapidly
decomposed by the action
of

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8
Fig. 3 Sensing layer structure
for organophosphorous
compounds

M.G. Valds et al.

OPH layer
Layers of
chitosan/TGA-

Layers of
OPH/TGAcapped
CdSe Clbs

Chitosan layer
Hydrophilic
support Sensing
architecture

sunlight
and
water.
Paraoxon, the neurotoxic
metabolite of OP insecticide
parathion,
exerts
acute
toxicity in target organisms
by
inhibition
of
acetylcholinesterase (AChE),
leading to the accumulation of
acetylcholine in cholinergic
synapses
and
overstimulation
of
the
cholinergic
system.
Recently, an interesting
optical nanosensor for the
detection of paraoxon has
been
presented
by
Constantine et al [52]. Layerby-layer films of chitosan,
organophosphorous hydrolase
(OPH) and thioglycolic acidcapped CdSe quantum dots
were arranged as a sensing
assembly (Fig. 3) for the
detection of paraoxon in
solution.
Luminescence
of
the
quantum dots immediately
decreased when exposed to !
AM solutions of the pesticide
due to the hydrolysis of the
organophosphorous
compound by OPH, thus
changing its conformation. Ji
et al. [53] employed this
same approach but using
CdSe-ZnS quantum dots
conjugat ed with OPH to

quantitatively
determine
paraoxon with a LOD as low
as 10-8mol L-1. The results
obtained verified that the
quenching of fluorescence
intensity of the OPH/QD
bioconjugate was due to
conformational changes in
the enzyme. Real sample
applications
of
these
devices is necessary to
demonstrate
their
applicability.
Another approach to
nano-sense paraoxon (and
also dichlorvos) is that
developed by Vamvakaki and
Chaniotakis [54] in which
the conventional biosensing
approach
for
pesticide
detection was employed:
inhibition of the activity of
the
enzyme
acetylcholinesterase
(AChE). In order to improve
the stability (over 50 days)
of the biosensor the enzyme
was encapsulated into the
internal nano-environment of
liposomes, that contained
porins incorporated within their
membranes. After preparation
of the liposome biosensing
aggregates (Fig. 4) a few L
of the solution were placed in
a

Fig. 4 Sensing liposome

nanocapsules for organopesticides [adapted from reference
54]

II

Pesticid
e

Enzyme

3/441/41,
41
* .1 7 *.

F-

substrat

Porin

Liposome
bilayer

.94 w1-

Active.
Enzyme

1
14
1

Inhibited
enzyme

11

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Fluoresce
nt

Analytical
10
nanotechnology for food analysis

fluorescence cuvette and the substrate acetylthiocoline was

added.
The enzymatic hydrolysis reaction then occured and
acetic acid was produced. The pH decrease was monitored
using pyramine, a pH sensitive fluorescent indicator
incorporated into the liposomes during construction of the
sensor. When the sensing nanocapsules were incubated
with sample solution containing the pesticide, enzyme
activity was inhibited and pH changes could be observed
fluorimetrically. The fluorescence intensity could then be
related to the OP pesticide concentration. Detection limits
for paraoxon and dichlorvos were found in the order of
10-10mo1 L-1. According to authors, these values were
better than those reported till then with similar pH
dependent fluorescence methods. Since the sensing nanocapsules could not distinguish between both pesticides,
total toxicity was determined in real drinking water samples
using standard addition of dichlorvos. Although the
preparation of the liposome-based nanosensor was rather
long, the results obtained were promising for further
applications in foodstuffs.
b) Parathion
Parathion is a non-systemic organophosphorus insecticide and acaricide with some fumigant action. Despite its
moderate toxicity, it is one of the most commonly used
pesticide and its highest permissible concentration set by
the European Union in foods was 0.05 mg Kg -1 [55]. M.
Wang and Z. Li [56] fabricated a Zr0 2/Au nano composite
film electrode and parathion was determined using squarewave voltammetry. A pre-adsorption step of the parathion
on the electrode was carried out before instrumental
determination. The nano film showed strong affinity to
parathion and the redox behaviour of the pesticide on the
film electrode was the typical reported. LOD was reported
to be 3 ng mL -1 and RSD was less than 5%. No food
samples were analysed.
c) Amitrole
Amitrole is a herbicide which has been used since the
1950s to control a wide range of weeds and grasses along
roadsides, in vineyards, in orchards and in other applications. Amitrole is rapidly degraded in the environment, but
occupational exposure may occur during its production and
application. In order to protect public health, a maximum
residue limit of 0.01 mg Kg-1 amitrol in foods has been
established by the European Union regulation [55].
Square-wave voltammetry was used by Siswana et al
[57], in the determination of amitrole employing a carbon
paste electrode modified with Fe(II)-phthalocyanine (FePh)
nanoparticles. Authors studied factors such as pH, loading
and presence of electrolytes, as well as the mechanism of
the interaction among nanoparticles and amitrole, in which
a redox process catalyzed by FePh was involved. The

M.G. Valds et 11
al.9

stability and selectivity of the nanoparticle modified

electrode seemed to be promising. The analysis of water
samples spiked with the herbicide demonstrated a 99.6% of
recovery and the reported LOD was 3.6 nmol L-1.
Trimethylamine (TMA) and biogenic amines
A remarkable feature of the post-mortem chemical changes
which take place in fish muscle is the increase in the level
of volatile amine compounds, which is strongly influenced
by conditions and duration of storage. TMA, responsible
for the characteristic smell of fish, is a metabolite of
trimethylamine oxide, a product of the metabolic route for
nitrogen removal. The concentration of TMA is a key
parameter for fish quality evaluation.
Two different approaches have been developed by Zheng
et al. [58] and by Zhang and Zhang [59] to determine TMA,
using different nano materials. Zheng et al. [58] developed
a polyaniline-TiO2 nano composited that was deposited on
the electrode of a quartz crystal in order to implement a
QCM sensor. Authors concluded that the analytical performance of this approach was not good enough and need
further improvement for practical applications. On the other
hand, Zhang and Zhang [59] synthesized SnO 2 nanoparticles and ZnO microrods that were mixed with an
organic binder to form pastes. The resulting pastes were
coated on Al203 tubes with a pair of Au electrodes attached
with Pt wires. Finally, the electrodes were fixed on a circuit
for measurement. A stationary state gas distribution method
was used for testing gas-sensing properties. The resistance
of the sensor in air or in a sample gas was measured by
monitoring the out potential. This sensing approach allowed
to determine as low as 1 in mL -1 of gaseous TMA with the
advantages of high selectivity, excellent sensitivity, strong
stability and prompt response/recovery. The semiconductor
gas sensor used 330 as working temperature and practical
application was demonstrated by introducing the vapours
from a dead Crucian fish at different storage times. The
resistance of the nanocomposite sensor showed a continuous and accelerating decrease with the storage time,
corresponding to the deterioration process of the dead fish.

H. Tang and co-workers [60] also employed nano

particles of surfactant-modified ZnO to determine TMA.
Polyvinylpyrrolidone/ZnO composite films were fabricated
on the top of Al electrodes by spin coating and the gas
sensing behaviour of the nanosensor was measured at room
temperature using a commercial measuring unit. The TMA
vapour was injected in N2 and the change in current
recorded. This sensor exhibited good selectivity and rapid
response being the detection limit lower than 0.4 in mL -1
TMA, thus improving the approach of Zhang and Zhang
[59] sensor. However, no real samples were analysed. Both,
Zhang and Zhang [59] and H. Tang et al. [60], discussed
the mechanism responsible for the enhancing in gas sensing

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properties as due to improvements in crystallization of

metal oxide nanoparticles which increased the electron-hole
transport, i.e. the working current.
Biogenic amines are formed during metabolic processes in
all living organisms and, therefore, they are present in foods.
Some of these amines are considered responsible of food
poisoning and most analytical methods for their assay in food
samples require a derivatization and a separation step.
Upon optimization of parameters, Ghanbari et al [61]
synthesized polypyrrole (PPy) nanofibers by electropolymerization of pyrrole unto a Pt disc electrode using normal
pulse voltammetry. Various parameters were also optimized
for the physi-sorption of double stranded DNA on the
nanofiber film and the resulting material was characterized.
The DNA/PPy sensor was then transferred to a spermidine
(SPD) solution for differential pulse voltammetry. Authors
showed that when DNA was sorbed unto the nanofiber
film-coated Pt electrode, the SPD/DNA interaction was
signalled by the oxidation peak of guanine present in DNA.
As SPD concentration increased (0.02-1.0 ilmol L -1), the
guanine peak current dropped linearly. A comparison was
made of the analytical performance of this sensor with other
reported methods for SPD observing that the LOD as well
as the linear range was much improved when the nanobased biosensor was used. No real samples were analyzed
for SPD.

Ethylene
Some foods, particularly fruits and vegetables, are able to
produce ethylene, a gas that can accelerate ripening,
softening, senescence and can induce a range of physiological disorders in produce. Precise and continuous
ethylene monitoring during fruit conservation is of interest
not only because low ethylene concentrations are produced
by the fruit itself and are indicative of its ripeness, but also
because ethylene may be externally added when ripeness or
degreening of the product must be promoted. Ethylene
continuous monitoring allows storage/distribution centres to
market fruit in optimal quality and safety conditions.
Ethylene was sensed by Pimtong-Ngam et al [62] using
a W03-SnO2 binary oxide with uniform distribution of
nano WO3 added to a SnO2 particle-based material. After
preparation, the sensor was placed in the middle of a quartz
tube inserted in a home made furnace and, after stabilization, ethylene gas was introduced into the chamber, flowing
constantly during 10-20 min at 300C. Conductivity measurements for concentrations between 2 and 8 in mL -1
showed a linear response. Analytical performance characteristics were poorly evaluated and no real samples were
analyzed.
Hypoxanthine
After the dead of a fish, ATP is decomposed in uric acid by

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a series of catabolic enzymes. Hypoxanthine, that imparts

the bitter "off-taste" in fish, is regarded as the major
catabolite of adenosine triphosphate (ATP) and it is a useful
freshness indicator because of its gradual accumulation in
flatfish and can be used as a measure of the duration of
icing. Consequently, simple and rapid methods for hypoxanthine determination are mandatory in the food industry.
A glassy carbon paste electrode based on xanthine
oxidase (XOD) and gold nanoparticles was prepared by
Cubukcu at al [63] for the chronoamperometic determination of hypoxanthine in tuna fish samples. The electrode
was prepared by mixing glassy carbon spherical powder,
XOD and gold nanoparticles. Authors showed that the
presence of the latter facilitated the enzymatic reactions, on
which sensor response was based:
XOD

Hypoxanthine + 02 -> Xanthine + H202

Xanthine + 02

OD

-> Uric acid + H202

The analytical characteristics were determined, but no

LOD was reported. A canned tuna fish sample was divided in
two parts in order to compare the results in the fresh portion
and in the "decomposed" 15 days-old portion. Hypoxanthine
standard was spiked in both portions and after analysis,
recoveries of 102.5 and 106% were found in the fresh and the
decomposed samples, respectively. Authors attributed the
higher recovery value for the decomposed sample to
interferences of other compounds caused by the microbial
contamination. Although the test with positive samples
indicated the potential analytical use of this nano based
sensor, the system requires more analyses of spiked and
incurred samples and a validation with reference methods.

Determination of hypoxanthine real sample foods was also

investigated by Agui et al [64] using an amperometric
biosensor based on immobilized XOD on carbon paste
electrodes modified with gold nanoparticles. The nano
sensing device was constructed by immersing a carbon paste
electrode (CPE) into a HAuCl4 solution for the electrodeposition of gold nanoparticles by applying a constant voltage.
The enzyme was then immobilized by coating the nanoAuCPE modified electrode with an XOD solution. Standard
curves for detection of hypoxanthine operating at +600 mV,
demonstrated a LOD of 1 x 10-7mol L-1, a quantifiable
range of 5 x 10-7-10-5 molL-1 and a useful long lifetime
(15 days of continuous use) of this nano-based sensor. The
biosensor was used for the amperometric determination of
hypoxanthine in 7 day-stored samples of sardines and
chicken meat. Both spiked and non spiked samples with
hypoxanthine were analyzed. Recoveries of spiked samples
were excellent while hypoxanthine was also detected in the
non spiked samples. Although the configuration of this
biosensor was different from that of Cubukcu at al [63], both
authors demonstrated that the AuNPs catalyze the electro-

chemical oxidation of the enzyme reaction products.

Polyphenol index
Polyphenols, secondary plant metabolites, are of great
importance for the food and drink products derived from
plants, not only because these compounds are responsible
for their organoleptic characteristics but also because their
biological activities including vasodilatatory, antiinflammatory and prevention of degenerative diseases such
as cancer and cardiovascular diseases, have been reported.
Wines contain a wide range of polyphenols that include
phenolic acids, flavonols (e.g. quercetin and myricetin),
flavan-3-ols (e.g. catechin and epicatechin), as well as
polymers of the latter (procyanidins and anthocyanins) that
are the pigments responsible for the colour of red wines.
The nutritional value of these phenolic components is due
to their antioxidant power, of special interest in red wines.
An electrochemical sensor based on the immobilization of
tyrosinase onto a glassy carbon electrode modified with
electrodeposited gold nanoparticles was recently developed
[65]. The enzyme catalyzed the oxidation of the phenol group
to o-quinone and the AuNP acted as nanoscale electrodes that
electrically communicated the enzyme with the bulk electrode. The electrochemical reduction of the quinones was
used to monitor the reaction. The practical usefulness of the
sensor was evaluated by estimating the "pool" of polyphenols
in red and white wines. Sample analysis was performed using
a standard addition method (caffeic acid as standard) and
results compared with a spectrophotometric method [66].
Although analytical characteristics of the sensor were good,
huge differences between the methods were obtained when
six wines were analyzed. A satisfactory explanation for those
differences was not provided.

Glucose
D-glucose occurs widely distributed in the plant and animal
kingdoms. The determination of glucose is a widespread
analytical task carried out in clinical and biochemical
laboratories as well as in the food industry. Glucose
biosensors account approximately 85% of the current world
market for biosensors [67], not only because the diabetes
represents one of main pathologies in the western world
that demands for the development of new methodologies
for a simple, rapid and reliable estimation of glycemia in
diabetic patients but also for the importance of glucose
determination in the food industry for quality control
purposes and in fermentation processes.

AuNP-CNT-Teflon electrode allowed the preparation of a

mediatorless GOx biosensor that combined the capabilities
of the CNTs and gold nanoparticles with the advantageous
properties of Teflon composite electrodes. A detection limit
of 17 pimol L-1 glucose and a useful sensor lifetime of
3 months (storage stability) were reported. The analytical
performance of the biosensor was compared that of a GOxCNT-Teflon one (absence of AuNPs) and results demonstrated that the presence of AuNP increased GOx activity
and stabilized its immobilization. The G0x-AuNP-CNTTeflon sensor was then applied to glucose determination in
sport beverages by the standard addition method. Comparison of results with those of a spectrophotometric flow
injection technique [69] that used immobilized GOD on a
Controlled Pore Glass reactor gave good correlation.
Recovery studies were also done on three of the five
samples analyzed and results were in the 98-105% range.
Q. Liu et al [70] reported a similar approach to glucose
sensing using quantum dots. A glass carbon electrode was
modified with CdTe QDs and CNT. This CdTe/CNTs
electrode was prepared by first mixing CdTe QDs, CNTs,
Nafion, and glucose oxidase (GOD) in appropriate amounts
and then modifying this mixture on the glass carbon
electrode (GC). The direct electrochemistry of GOD on the
Nafion-CdTe-GOD modified electrode was investigated and
cyclic voltammograms demonstrated that the direct electron
transfer of GOD was achieved through the incorporation of
GOD finto the Nafion-CdTe film. Authors claimed that CdTe
QDs played an important role in facilitating the electron
transfer between the enzymes and the electrode surface,
probably due to a high charge detaching efficiency of
quantum dots resulting from their quantum size effect along
with a favorable orientation of GOD molecules in the
composite film. On the other hand, CNTs acted as nanowiring of metalloenzymes, so that combining CNTs with
QDs resulted in a synergistic effect that enhanced the
analytical performance of the biosensing platform. A linear
relationship between glucose concentration and current
values was obtained up to 0.7 mmol L-1, but the detection
limit was higher than those reported in the two previous
papers. Although authors did not apply this biosensor to any
particular sample, it was recommended for food analysis.

Glucose was also determined by J. Li [71] and co-workers

in beer. GOx was immobilized on a film composed of AuNPdimethylformamide (DMF) and the ionic liquid 1-octy1-3methylimidazolium hexafluorophosphate (OMIMPF6). The
mixture was deposited on a glassy carbon electrode and the
film was allowed to dry at room temperature for 8 h. Well

A gold nanoparticles (AuNP)-carbon nanotubes (CNT)

composite electrode using Teflon as non-conducting binder
has been reported by Pingarrn et al [68] for the
determination of glucose in beverages. The authors claim
that incorporation of glucose oxidase (GOx) to the resulting

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12

defined peak currents were obtained only if all components

were present. The role of each component was justified as
follows. The nanometric edges of gold AuNPs protruded into
the protein, allowing the distance between the electrode and
the GOx redox center to decrease. On the other hand, the
native structure of GOx was preserved by hydrogen bonds
with DMF while the presence of OMHV1PF 6 improved the
stability and bioactivity of GOx. A linear relationship between
glucose concentration and peak current was reported in a
range of 10-7 to 2x 1 0-511101 L-1 glucose. The sensor was
tested in spiked beer samples and a 99% average recovery was
obtained. The proposed sensor was judged feasible for glucose
determination not only in beers but also in plasma samples;
however, no detection limit was reported and a comparison
with a confirmatory reference method should be performed.

Folic acid
In order to improve their nutritional value, many times food
must be fortified with certain components. Such is the case
of folic acid which is a soluble vitamin whose deficiency
results in anemia and that is especially important during
periods of rapid cell division and growth such as infancy
and pregnancy. Several health agencies [72, 73] have made
it mandatory to fortify certain food products with this
compound. In order to analyze folic acid in fortified foods,
F. Xiao et al [74] have prepared a folic acid sensor using a
single walled carbon nanotube paste coated glassy electrode
using the ionic liquid OMIMPF6 as binder. The SWCNTs
were mixed with the binder in a 1:1 ratio and a proper
amount of the mixture was transferred to the glassy carbon
electrode surface. The OMIMPF6-SWCNT paste modified
glassy carbon electrode was installed in the electrochemical
cell. Voltammetric measurements were made after an
accumulation time on open circuit for 6 min. Various
factors were studied for optimization of the sensor and the
LOD was reported as low as 1.0x10 -9mol L-1 which,
according to authors, was lower than that obtained by other
conventional electrochemical methods. Authors considered
there was a synergistic effect between the CNT and the
binder which increased the response of folic acid. Food
samples were pre-treated as necessary and folic acid
determined. Folic acid was determined in wheat flour, fruit
juice and milk samples. Recoveries ranged between 93108%, when the standard addition method was performed.

Wang et al. [75] have also reported the voltammetric

determination of folic acid in food supplements (tablets)
using a single-wall CNT film electrode. The system
demonstrated to be highly sensitive, selective and stable.
The reduction peak current was found to be linear over the
range 1 x10-8mol L-1-1 x10-4mol L1 folic acid. The LOD
was reported to be 1 x10 -9mol L-1 alter 5 min accumulation.
Although authors claimed that the CNT film electrode may
be a good electrochemical sensor for the direct measurements

4 Springer

M.G. Valds et al.

of folic acid, application to determine it in tablets (dietary

supplement) was performed by a standard addition method
and recoveries ranging from 98.03% to 102.7% were found.
Melatonin
Studies suggest that sleep disorders affect 50-70 million
Americans, representing approximately 20% of the population
[76]. In addition to the adult population, difficulties initiating
and maintaining sleep are very common in children,
affecting about 15-25% of this population [76]. Melatonin
(N-acetyl-5-methoxytryptamine) is a neurohormone that is
primarily produced by the pineal gland, located behind the
third ventricle in the brain. Melatonin is believed to be
responsible for the synchrony of the circadian rhythm in
humans, an interna! 24-hour time-keeping system that
modulates sleep patterns with day and night. Other functions
for melatonin have been suggested, including scavenging of
free radicals, neuroprotection, anticonvulsant efficacy and
strengthening immunity. There are natural plant sources of
melatonin (feverfew leafs, flowers of St. Jonh's wort, white
mustard seeds, etc) but commercially available melatonin (as
food supplement) is isolated from the pineal gland of beef
cattle or chemically synthesized.
Qu et al. [77] developed a multi-wall carbon nanotube
film coated glassy carbon electrode for melatonin determination. Under optimal conditions, a linear response of
melatonin was obtained in the range from 8 x 10-8 to 1 x
10-5M, being the LOD 2 x10-8mol L-1 melatonin. A
relative standard deviation of 4.9% for 10 parallel detections
of 1 x10-6mol L-1 melatonin was reported. The sensor was
applied to the voltammetric determination of melatonin in
Naobaijin capsules, a salable sanitarian foodstuff. Recovery
values ranged between 99.2% and 103.2% and consistent
results were obtained when compared with those obtained by
a HPLC method for melatonin determination.

Cyanide
Cyanides may be occasionally found in drinking-water,
primarily as a consequence of industrial contamination. The
highest concentration of cyanide allowed in drinking water
by the US EPA (Environmental Protection Agency) is
200g L-1 [78], while the directive 1998/83 of the
European Union on the quality of drinking water sets a
lower limit of 50 in L -1 [79]. The maximum amount of
cyanides allowed in mineral waters according to directive
2003/40/E.U. is 70 in L-1 [80].
There are also more than 2500 species of plants that
produce cyanoglycosides that yield cyanide (cyanogenesis)
following their enzymatic breakdown. This combination of
cyanoglycoside and hydrolytic enzyme is the means by

Analytical nanotechnology for food analysis

which cyanogenic plants are protected against predators.

The major food sources of cyanogenic glucosides include
many important crop species such as sorghum, almonds,
lima beans (nondomesticated) and white clover. The most
agronomically important of the cyanogenic crops, however,
is the tropical root crop cassava (Manihot esculenta
Crantz). It is the third most important food source in the
tropics after rice and maize and is the staple food of about
600 million people. Consumption of cassava and its
products that contain large amounts of cyanogens may
cause cyanide poisoning with symptoms of vomiting,
nausea, dizziness, stomach pains, weakness, headache and
diarrhea and occasionally death. The lethal dose range for
humans of hydrogen cyanide taken by mouth for a 60 Kg
adult amounts to 30-210 mg of HCN [81].
Although conventional techniques such as spectrophotometry give satisfactory analytical results for cyanide
determination in waters [82] and cyanogenic foods [83],
new sensors for cheaper and faster on-site analysis are
being developed. An example is given by H. Sun et al [84]
that developed a cyanide sensor based on piezoelectric
quartz crystal (PQC) by depositing photochemicallygenerated (illumination 1 h at 254 nm) nano sized silver
particles on TiO2 coated on the electrode surface of the
PQC. The AgNPs were generated by illumination of a
silver nitrate solution during 1 h at 254 nm. According to
the authors, the freshly produced AgNPs on the TiO 2 thin
film, interacted strongly with cyanide ions and sensor
response was much improved in comparison with a bulk
silver-coated PQC. The mechanism of response upon
cyanide interaction was based on the following reaction:
4Ag + 02 + 8CN- + 2H20 > 4Ag(CN)2 + 40HThe gradual removal of Ag from the electrode results in
an increase of frequency. Sensor performance was evaluated under different experimental conditions. The analytical
characteristics of the sensor and factors affecting its
application were also studied. Drinking water samples were
spiked with trace amounts of NaCN and recoveries
obtained ranged from 94 to 108%. The LOD reported
(2.2 in L-1) was said to be more than thirty times lower
than the value established for CN -, by the World Health
Organization in this type of samples.

Other applications of nano materials in the analysis

of species of interest in the food industry

Nano-based noses and tongues

It has been estimated that humans are able to detect as

13

chemical structures are perceived as different odours. Even,

humans have the ability to distinguish enantiomers, e.g.
(S)-(-)-limonene has a harsh, turpentine-like, odour, while
(R)-(+)-limonene has a fresh citrus, orange-like odour. The
lowest concentration of an odour threshold can be less
than one part per trillion in air [85]. Artificial systems
mimicking the natural olfaction sense were described in
early 1980s, while the term 'electronic nose', or e-nose,
was defined around 1994 by Gardner and Bartlett [86].
Typically, e-noses consist of four components: a) an array
of thin or thick film semiconductor sensors, with partially
overlapping sensitivities which change their conductance
when exposed to gases, b) an electronic circuit, c) a
sampling system and d) data analysis software [86].
Although every sensor in an array may respond to a given
chemical, these responses will usually be different, so that
an odour stimulus generates a characteristic fingerprint
from the array of sensors. The pattern-recognizer compares
the pattern of the measurements to stored patterns for
known materials [87]. In summary, the e-noses recognize a
fingerprint, that is the global information of the sample to
be classified. E-noses are moving into food, beverage,
medical, and environmental applications. In particular, enoses (and e-tongues) are being used in the laboratory to
develop new foods, beverages, and pharmaceuticals and in
production plants to monitor the quality of products being
produced.

Modified nanoparticles (nanosensors), can be used to

create tiny sensors with abilities to detect odours and tastes.
Arrays of these nanosensors are able to detect molecules on
the order of one part per million, sniffing molecules out of
the air or taste them in a liquid, suggesting applications in
foods and food industry. So, during the last few years, enoses have been developed as an alternative method to
perform aromatic analysis giving an overall response to a
mixture without identifying the single components [88].
a) Vinegars characterization
Although its major components are water and acetic
acid, vinegar is characterized by a spectrum of different
organoleptic features that is given by the around of 100
aromatic chemical compounds. This complexity makes
chemical analysis a difficult task. In order to afford this
challenge, Q. Zhang et al [89] have developed an electronic
nose comprised of nine thick film gas sensor prepared from
ZnO nanoparticles doped with metal oxides or metals. The
response of the array to 17 different vinegar samples was
obtained by injecting each sample into the measurement
chamber and vaporizing. The gas signal in each case was
obtained by expressing the resistance of the sensor in air
and in the gas sample. The results were then studied by
principal component analysis (to investigate the presence of
classes among the samples), cluster analysis (to examine

many as ten thousand different odorants: subtle changes of

1 Springer

14

the sensorial data and test the relationship between various

vinegars) and a neural network was used for classification.
This work was a first step, according to authors, to the
development of a commercial electronic nose for Chinese
vinegar characterization [90].
b) Bacteria characteristic vapours
As can be seen from what has been summarized up to here,
most of nano-sensors for pathogens were based, in one way or
another, in an antibody/antigen interaction for their detection.
There are other innovative possibilities for recognition. So,
Arshak et al [91] have proposed an array of conducting
polymer nanocomposite sensors to detect the gases and
odours produced by the microorganisms during food
spoilage. These gas sensors, containing carbon black nanoparticles and polyaniline, detected and identified the pathogens through the production of individual response patterns,
characteristic of each one. Four composite sensor materials
were tested with the gases produced by Salmonella spp.,
Bacillus cereus and Vibrio parahaemolyticus. The voltage of
the sensors varied when exposed to bacteria vapours and
investigations were continued to verify response mechanisms. According to the authors, the array showed potential
for on site identification of food borne pathogens where they
could be interfaced with handheld devices to quantify gas
emissions of contaminated food samples.

The electronic tongues are now coming to the forefront

having been shown to be more robust and reproducible than
their e-nose counterparts. Electronic tongues are sensor
devices capable to determine the composition and to
discriminate food tastes of different nature. Recently, a
review on amperometric electronic tongues for food
analysis has been published by M. Scampicchio et al [92]
in which descriptions of different types of amperometric
sensors used in electronic tongues, the processing of sensor
responses by means of pattern recognition techniques and
the advantages of e-tongues for the evaluation of food taste
can be found. It is worth noting that authors highlighted the
fact that, in spite of the literature is rich in reviews of
sensors based on nanomaterials (nanoparticles, nanoconducting polymers), no amperometric nano-based etongues have been described yet. The application of
nanomaterials to the development of e-tongues is new but
has a considerable potential. For example, G. Pioggia et al
[93] have developed a composite CNT/polymer sensor for
use in an impedimetric e-tongue and tested its response
with five compounds with different chemical characteristics
able to elicit different kinds of gustative perceptions,
representing five classic tastes in foods. Results demonstrated that the system was suitable for concentration
discrimination of the compounds, being the sensor reproducible and highly sensitive to all tastes except sweet. c)
Prion proteins

4 Springer

M.G. Valds et al.

Prion proteins (PrP), infectious particles that lack nucleic

acid, cause neurodegenerative diseases that are known as
bovine spongiform encephalopathy (BSE) in cattle,
Creutzfeldt-Jacob disease (CJD) in humans, scrapie (SC)
in sheep, transmissible mink encephalopathy (TME) in
mink, chronic wasting disease (CWD) in mule deer and elk
[94]. The normal cellular PrP, denoted as PrP e is converted
into infectious forms (designated as PrP BSE, prpCJD, prpSC,
PrPTIvIE and PrPcwD) through a process whereby a portion of
it's oc-helical and coils structure is refolded into (3-sheet.
This modification is accompanied by drastic changes in the
physicochemical properties of the PrP: aggregation, insolubility, protease digestion resistance [94]. Prion diseases
may be caused not only by genetic or sporadic disorders but
also by infectious ones through the food chain. In order to
improve food safety, the development of reliable analytical
methods would be beneficial to screen all the animals for
prion disease using ante mortem tests, regardless of the
presence of symptoms.
Although it is not an e-nose or an e-tongue, Craighead et al.
[95] has recently developed an array of resonant sensors to
detect prion proteins using secondary mass labelling. Arrays
of low-stress silicon nitride as resonators were used as
sensing platform for PrP detection. The surfaces were
functionalized via silanization and glutaraldehyde chemistry.
Primary monoclonal antibodies against amino acids 23-237
of bovine prion protein were then immobilized and a
sandwich assay was employed for the detection of PrP using
biotinylated-secondary antibodies (against amino acids 123136 and 140-160 of bovine prion protein). In order to
enhance the frequency shift for detection of PrP, streptavidin
conjugated were used thus overcoming limitations of
resonant sensors at low analyte concentrations. Authors
observed that a) without the use of the secondary antibody,
PrP was not detected at concentrations below 20 in mL-1, b)
in the presence of secondary antibodies the analytical
sensitivity was improved to 2 in mL -1 and c) with the use
of functionalized nanoparticles, the sensitivity improved an
additional 3 orders of magnitude to 2 ng mL-1. Although the
device has not been applied to real samples, authors claimed
it may be suitable to develop ante mortem tests to directly
detect PrP in body fluids.

Nanoparticles in solution
Nanomaterials have not only been used in the development
of sensing devices; they have also been employed as part of
diverse analytical methods where they can play very
different roles. The following are only some recent
examples of these other applications.

Analytical nanotechnology for food analysis

Diaz-Garcia et al [96] have used gold nanoparticles and

europium ions in the analysis of amino acids, lysine in
particular, in syrups used as a dietary supplement. The
interaction of mercaptoundecanoic acid capped AuNPs with
Eu3 ions resulted in AuNPs surface plasmon spectral
changes. At the same time, the europium ions (complexed
with acetate ions in buffer solution) fluorescence at 590 and
620 nm was quenched. Non radiative energy transfer from
excited Eu(III) to the nearby AuNPs through binding with
the S(CH2)10C00- groups was proposed as decay channel
for the excited ions. On the other hand, it was observed that
the light scattering of the AuNPs/Eu 3 system was
enhanced upon addition of amino acids, particularly when
lysine was added. Interference from other amino acids was
marginal. Interplasmon coupling phenomena suggested that
the functionalized NPs were brought together upon addition
of the lysine. The analytical performance characteristics of
this nano-chemosensing approach as well as its application
in a dietary supplement syrup demonstrated the analytical
potential of the approach.
On the other hand, Dasary et al [97] used AuNPs
without functionalization and, in this case, their interaction
with Eu3 ions produced an enhancement in their fluorescence at 615 nm. Authors first explained the surface
enhanced fluorescence observed, and then the quenching
mechanism that was produced in the presence of an
organophosphorus pesticides (OPA) (Fig. 5).
Since the binding constant of the europium ion with
OPA was much higher than with the AuNP, Eu 3 was
released from the nanoparticle surface, resulting in a
fluorescence change depending on OPA concentration.
Although linearity was 1-25 punol L -1, no other analytical
characteristics were given and apparently no selectivity
studies were performed. No real samples were assayed.
Fig. 5 Competitive assay for

Magnetic nanoparticles
havepestices
also used for separations in
organophosphorus
some assays. For
Amagliani [98] used paramagusingexample,
gold nanoparticles
[adapted
frommagnetite
Ref. 97] beads or agarose based
netic nanoparticles
(silica
paramagnetic beads derivatized with diethylaminoethyl) for
the isolation of bacterial DNA (Listeria monocytogenes)
directly from the milk and subsequent PCR analysis with
selective primers for the listeriolysin O gene. Yang [99], on
the other hand, used carboxylic acid functionalized iron
oxide magnetic nanoparticles, modified by covalently
bound rabbit anti-Listeria monocytogenes, for separation
in order to screen the presence of the bacteria in the
samples with real-time PCR analysis. In both works, the
sensitivity of the corresponding assay was reported as much
higher than those using commercially available separation
means.
Quantum dots are yet another type of nanoparticles
employed as fluorescent labels by several authors for the
detection ofListeria monocytogenes, Salmonella typhimurium
and Escherichia coli 0157:H7. So, an anti-Intemalin B (In1B)
polyclonal antibody and a recombinant antibody fragment
were used by Tully et al [43] to develop a fluorescence
competitive assay for Listeria monocytogenes cell surface

15

proteins, based on QD labelling. According to the authors, the

preliminary results obtained were not as sensitive as ELISAbased assays due to differences in the QD-conjugate
preparations.
Y. Su [100] demonstrated the possibility of using 605 nm
quantum dots as fluorescence labels in the detection of
Escherichia coli 0157:H7. Later, Yang and Li [101] used
QDs of different sizes for the simultaneous detection of
Salmonella typhimurium and Escherichia coli 0157:H7.
These QDs were conjugated with the corresponding antibodies and, in this way, selective labels of different colours
for individual bacteria were used for simultaneous detection. The detection scheme, where target bacteria were
separated using specific antibody coated magnetic beads, is
presented in Fig. 6.
The reported LOD (104cfu mL-1) was comparable to
other single bacteria methods. More recently, Y. Liu et al
[102] used a similar procedure with magnetic beads to
separate target DNA by probe hybridization, using multi
layers of QDs to amplify fluorescence signal in the
detection of Escherichia coli 0157:H7. Although all these
authors highlighted the importance of determining the
pathogens in food, none applied their developed assays to
real sample analysis.

Low
europium
fluorescenc

\ -t

Springer

16

M.G. Valds et al.

Fig. 6 Schematic immunoassay

Streptavidin
conjugated QD

protocol for detection of bacteria. S. Typhimurium cells and

E. coli 0157:H7 cells, using
magnetic beads for separation
and QDantibody conjugates as
fluorescence labels [Adapted
from reference 100]

Biotin-labeled
antibody

Antibody coated
tr.11- QDsconjugated
_____7___...antibodies )

magnetic beads

Separatio
n

E-coli cell

Interferent
5.Typhimurium

Wast
e

Nano teststrips
Gold
nanoparticl
es
were
also
employed
for
an
immuno
assay
for
the
detection of
aflatoxin
B1 (AFB1)
in
foods
[103].
Colloidal
AuNP were
labelled
with
polyclonal
antibody
specific to
AFB1 and
an AFB1BSA
conjugate
was used as
the
competitive
antigen.
Test strips
(Fig.
7)
were
prepared
that
contained 4

component
s: sample
application
pad,
conjugate
release
pad,
an
analytical
membrane
and
an
absorbent
pad.
The
conjugate
pad
was
coated with
the
modified
gold probe
particles
and
upon
AFB
1
standards
or sample
extracts
were added
to
the
bottom of
the
strip,
reaction
between
conjugate
and analyte
took place.
The
analyte and
immobilized
AFB1-BSA

.0
...
Excitatio
n X = 395
nm

Emission
525
nm
X=
705 nm

k=

competed
for binding
to the gold
antibody
probe and
as soon as
the
solution
reached
the top of
the strip, a
color
signal
appeared.
Quantitation
was
obtained
measuring
the optical
density at
540 nm. In
the absence
of
AFB1,
the binding
of
the
immunogol
d labelled
antibody
with
the
solid phase
AFB-BSA
developed a
red band,
the colocar
of
which
disappeared
when
the
analyte was
present. A
standard
addition
process was
performed
in
the
analysis of
food
samples
(rice, corn
and wheat).
AFB
1
recoveries
and
the
variation
coefficients
differed
greatly
between the
different
types
of

samples.
LOD given
was ca. 2in
mL-1
AFB1.
Good
agreement
between
results
were
obtained
when
samples
were
analyzed
using
an
ELISA kit
test.

Conclusion
s

While there
are some
niche
applications
where
nanotechnology has
penetrated
the food
market
(packing
materials,
antibacterial
coatings,
supplement
delivery
systems),
the major
impact in
food
analysis
will be at
least
a
decade
away. It is
evident that
the
food
industry is
an
open
market for
the
analytical
application

of
nano
materials.
Since many
important
chemical
and
physical
interactions
on
which
sensing
relies are
governed
by surface
and
surface
properties,
nanoscale
materials
offer
a
tremendous
potential
due to their
large
surface area
for a given
volume. In
fact, almost
every type
of
nanomateria
l has been
employed to
develop
sensors,
mostly
biosensors,
but among
the
most
11Z Springer

popular are
gold

nanoparticl
es
and
carbon
nanotubes.
Scarce
works have
been
reported in
reference to
analytical
methods no
including
sensors.
On
the
other hand,
the
most
extended
means
of
sensor
transductio
n
is
electrochem
ical,
especially
amperometr
y and cyclic
voltammetry
. Quantum
dots are the
most
applied
._
A ds
o rb
en t
pa d

Fig. 7
Diagram of a
nano-based
immunoassay
test strip

4- Control
line Test
line
Gold
layer
Filter
pad
Sample

Analytical nanotechnology for food analysis

optical nanoparticles, particularly as fluorescent markers in

assays of analytes of concern in foodstuffs. Many have been
the (bio)chemical procedures used by authors to develop the
recognition process, being the antigen-antibody approach the
most common.
Since only a moderate success has been made over the
last 10 years in the development of nano-based analytical
devices, researchers must continue to investigate strategies
to optimize the fabrication of nanosensors and nanomaterials to achieve improved analytical performance. As
it occurs frequently, most authors recommend their nanomethod or sensor for application in the food industry;
however, not only are there few real-world applications,
there are even very few applications to real samples. The
need of commercializing sensors and methods that can
rapidly screen analytes of interest in foodstuffs, at low
levels, with accuracy and precision, is a challenge for
nanotechnology and, although much has been researched,
much is still to be done for real world application of nano
scale technology to the food industry. Again, analytical
chemistry has to come out of the labs and go into real life,
especially with the surfacing of materials with such
excellent qualities as those shown by nano materials. Here
is the real challenge.

Acknowledgements Authors gratefully thank the University of

Oviedo (Spain) and the University of La Habana (Cuba) for financial
support (Research Agreement 2008).

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