Determining nanomaterials in food Cristina Blasco, Yolanda Pico ´ Nanotechnology has emerged as one of the most innovative technologies and has the potential to improve food quality and safety. However, there are a few studies demonstrating that nanomaterials (NMs) are not inherently benign. This review highlights some current applications of NMs in food, food additives and food-contact materials, and reviews analytical approaches suitable to address food-safety issues related to nanotechnology. We start with a preliminary discussion on the current regulatory situation with respect to nanotechnology in relation to foods. We cover sample preparation, imaging techniques (e.g., electron microscopy, scanning electron microscopy and X-ray micros- copy), separation methods (e.g., field-flow fractionation and chromatographic techniques) and detection or characterization techniques (e.g., light scattering, Raman spectroscopy and mass spectrometry). We also show the first applications of the analysis of NMs in food matrices. ª 2010 Elsevier Ltd. All rights reserved. Keywords: Consumer safety; Food; Food additives; Food analysis; Food matrix; Food packaging; Nanomaterial; Nanoparticle; Nanotechnology; Regulatory framework 1. Introduction A number of recent reports and reviews have identified current, short-term projected applications of nanomaterials (NMs) in the food industry, and for food and beverages [1–5]: (1) development of materials with novel functionality; (2) microscale and nanoscale processing; (3) new products development; and, (4) design of methods and instrumenta- tion for food safety and biosecurity. Fig. 1 identifies these areas of applica- tion in the food-processing chain, grouped by target area. A complex set of engi- neering and scientific challenges in the food and bioprocessing industry in man- ufacturing high-quality, safe food with efficient, sustainable resources can be solved through nanotechnology. Among emerging applications of nanotechnology in the food industry are: (1) bacteria identification and food-qual- ity monitoring using biosensors [6]; (2) intelligent, active, and smart food- packaging systems [7]; and, (3) nanoencapsulation of bioactive food compounds (e.g., micelles, liposomes, nanoemulsion, biopolymeric nano- particles, and cubosomes) [2,3]. Table 1 sets out some examples of NMs applied to food, divided into several cate- gories including food, food additives and food packaging, using many different types of materials [e.g., membrane, nanocapsule, nanoemulsion, liposomal nanovesicle, nanotube (NT), nanosphere, nanoceramic, nanoclay and nanowire]. According to a study from iRAP, Inc. [8], the total nano-enabled food and beverage packaging market in the year 2008 was $4.13bn, which grew in 2009 to $4.21bn and is forecast to grow to $7.3bn by 2014, at a compound annual growth rate of 11.65%. Active technology represents the largest share of the market, and will con- tinue to do so in 2014 with $4.35bn in sales, and the intelligent segment will grow to $2.47bn. The US NM market, which to- taled only $125m in 2000, is expected to reach $30bn by 2020, and packaging with nanotechnology is expected to grow at 11.65% from 2008 until 2013. While the majority of manufacturing and use of nanoscale materials is in USA, the European Union (EU), with its global share of the sector of around 30%, is not lagging far behind in this field [9]. Although the prospective beneficial effects of nanotechnologies are generally well described, studies assessing their po- tential toxicological effects and impacts are still limited [10,11]. However, the scientific community is concerned about this issue, and there is now a wider debate about the risks of the many manufactured NMs. Due to this, hundreds of in vitro Cristina Blasco, Yolanda Pico ´* Laboratori de Nutricio ´i Bromatologia, Facultat de Farma `cia, Universitat de Vale `ncia, Av. Vicent Andre ´s Estelle ´s s/n, 46100 Burjassot, Vale `ncia, Spain * Corresponding author. Tel. +34 96 3543092; Fax: +34 96 3544954; E-mail: [email protected]Trends Trends in Analytical Chemistry, Vol. 30, No. 1, 2011 84 0165-9936/$ - see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2010.08.010
Transcript
Trends Trends in Analytical Chemistry, Vol. 30, No. 1, 2011
Determining nanomaterials in foodCristina Blasco, Yolanda Pico
Nanotechnology has emerged as one of the most innovative technologies and has the potential to improve food quality and safety.
However, there are a few studies demonstrating that nanomaterials (NMs) are not inherently benign.
This review highlights some current applications of NMs in food, food additives and food-contact materials, and reviews
analytical approaches suitable to address food-safety issues related to nanotechnology.
We start with a preliminary discussion on the current regulatory situation with respect to nanotechnology in relation to foods.
We cover sample preparation, imaging techniques (e.g., electron microscopy, scanning electron microscopy and X-ray micros-
copy), separation methods (e.g., field-flow fractionation and chromatographic techniques) and detection or characterization
techniques (e.g., light scattering, Raman spectroscopy and mass spectrometry). We also show the first applications of the analysis
Anumberofrecentreportsand reviews haveidentified current, short-term projectedapplications of nanomaterials (NMs) in thefood industry, and for food and beverages[1–5]:(1) development of materials with novel
functionality;(2) microscale and nanoscale processing;(3) new products development; and,(4) design of methods and instrumenta-
tion for food safety and biosecurity.Fig. 1 identifies these areas of applica-
tion in the food-processing chain, groupedby target area. A complex set of engi-neering and scientific challenges in thefood and bioprocessing industry in man-ufacturing high-quality, safe food withefficient, sustainable resources can besolved through nanotechnology. Amongemerging applications of nanotechnologyin the food industry are:(1) bacteria identification and food-qual-
ity monitoring using biosensors [6];(2) intelligent, active, and smart food-
packaging systems [7]; and,(3) nanoencapsulation of bioactive food
compounds (e.g., micelles, liposomes,nanoemulsion, biopolymeric nano-particles, and cubosomes) [2,3].
Table 1 sets out some examples of NMsapplied to food, divided into several cate-gories including food, food additives and
0165-9936/$ - see front matter ª 2010 Elsev
food packaging, using many different typesof materials [e.g., membrane, nanocapsule,nanoemulsion, liposomal nanovesicle,nanotube (NT), nanosphere, nanoceramic,nanoclay and nanowire].
According to a study from iRAP, Inc. [8],the total nano-enabled food and beveragepackaging market in the year 2008 was$4.13bn, which grew in 2009 to $4.21bnand is forecast to grow to $7.3bn by 2014,at a compound annual growth rate of11.65%. Active technology represents thelargest share of the market, and will con-tinue to do so in 2014 with $4.35bn insales, and the intelligent segment will growto $2.47bn. The US NM market, which to-taled only $125m in 2000, is expected toreach $30bn by 2020, and packaging withnanotechnology is expected to grow at11.65% from 2008 until 2013. While themajority of manufacturing and use ofnanoscale materials is in USA, theEuropean Union (EU), with its global shareof the sector of around 30%, is not laggingfar behind in this field [9].
Although the prospective beneficialeffects of nanotechnologies are generallywell described, studies assessing their po-tential toxicological effects and impactsare still limited [10,11]. However, thescientific community is concerned aboutthis issue, and there is now a wider debateabout the risks of the many manufacturedNMs. Due to this, hundreds of in vitro
ier Ltd. All rights reserved. doi:10.1016/j.trac.2010.08.010
Figure 1. Nano applications in food and the food industry.
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toxicological studies have been reported, as well asnumerous reviews and perspectives [12–18]. Recentexamples in the literature show that engineered inor-ganic nanoparticles (NPs) and carbon nanostructuresmay incidentally or intentionally enter into contact withliving organisms, may disrupt normal activity and maylead to malfunctioning and diseases [19].
NMs are able to cross biological membranes and accesscells, tissues and organs that larger particles normallycannot [19]. NMs can also enter the blood stream viainhalation or ingestion, and some NMs penetrate the skin[20]. Then, once in the blood stream, NMs can betransported around the body and taken up by organs andtissues, including brain, heart, liver, kidneys, spleen,bone marrow and nervous system. Studies demonstratethe potential of NMs to cause DNA mutation and inducemajor structural damage to mitochondria, even resultingin cell death [21]. Size is a key factor in determining thepotential toxicity of a particle [22], but there are alsoother contributing aspects (e.g., chemical composition,morphology or shape, surface structure, surface charge,aggregation and solubility, and the presence or absenceof other chemical functional groups) [10].
It is difficult to generalize about health risks associatedwith exposure to NMs – each new NM must be individually
assessed, taking into account all material properties. As aconsequence, international agencies and governmentsare paying attention to the study of the fate, transport, andhealth effects of NMs in food and the environment. Severalreviews already present the latest research carried out toassess the risks of engineering NMs (ENMs) in the aquaticenvironment, including analytical methods and ecotox-icity assessments [23–28]. Outstandingly, the latest re-views, focusing on emerging contaminants in food and theenvironment, include NMs as one of the hottest topics inresearch today [29–35]. The prerequisite for toxicological,toxicokinetic, migration and exposure assessment is thedevelopment of analytical tools for detection and charac-terization of NPs in complex matrices [22].
Due to consumer safety, it is necessary to control thecontent of NMs in food [36]. To obtain this informa-tion, reliable quantitative methods of analysis arerequired to measure levels of NMs in a broad range ofmatrices. In food, there are natural NMs, intentionallyadded ENMs – derived from naturally occurring foodcomponents, or engineered using materials that are notendogenous to foods – and NMs resulting from con-tamination. Many food substances or ingredients havenanostructures in nature and are present at lm or nmin size:
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Table 1. Nanomaterials (NMs) in food, food additives and food packaging materials
Product name Manufacturer NMs Claim Web address or reference
Food packaging materialsDurethan KU 2-2601 Bayer Silica in a polymer-
based nanocompositeNanoparticles of silica in theplastic prevent the penetration ofoxygen and gas of the wrapping,extending the product shelf life
Nano zinc light catalyst Biodegradable after useCompostable to Europeanstandards EN13432Made from renewable andsustainable resources (non-GMcorn starch)Water dispersible, will notpollute local groundwatersystems or waterwaysIn use since 2002
http://www.physorg.com/news717488335.html
Constantia multifilmN-COAT
Constantia Multifilm Nanocomposite polymer A clear laminate withoutstanding gas-barrierproprieties developed primarilyfor the nuts, dry food and snackmarkets
http://www.constantia-multifilm.com/
DuPont Light Stabilizer210
Du Pont Nano TiO2 UV-protected plastic foodpackaging
NanoCoQ10 Pharmanex Nano coQ10 Nano technology to deliverhighly bioavailable coenzymeQ10, making it up to 10 timesmore bioavailable than otherforms of CoQ10
Fortified fruit juice High Vive.com 300 nm iron(SunActive Fe)
http://www.highwive.com/sunactiveiron.htm
Daily Vitamin Boost Jamba Juice Hawaii 300 nm iron(SunActive Fe)
22 essential vitamins andminerals and 100%, or more ofyour daily needs of 18 of them!
http://jambajuicehawaii.com/vita-boost.asp
Oat ChocolateNutritional Drink Mix
Toddler Health 300 nm iron (SunActiveFe)
Toddler health is an all-naturalbalanced nutritional drink forchildren from 13 months to5 years. One serving of ToddlerHealth helps little ones meettheir daily requirements forvitamins, minerals and protein
http://www.toddlehealth.net/OatChocolate.php
Oat Vanilla NutritionalDrink Mix
Toddler Health 300 nm iron (SunActiveFe)
Toddler health is an all-naturalbalanced nutritional drink forchildren from 13 months to5 years. One serving of ToddlerHealth helps little ones meettheir daily requirements forvitamins, minerals and protein
http://www.toddlehealth.net/OatChocolate.php
Canola Active oil Shemen Nano-sizedselfassembled structuredliquid micelles
http://www.shemen.co.il
Trends in Analytical Chemistry, Vol. 30, No. 1, 2011 Trends
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(1) food proteins, which are globular particles of 10s to100s of nm in size, are true NPs;
(2) linear polysaccharides with one-dimensional nano-structures are less than 1 nm in thickness; and,
(3) starch polysaccharides have small 3-D crystallinenanostructures that are only 10s of nm in thickness.
Although natural NMs in food are not consideredwithin the scope of this review, they contribute to thecomplexity of the analysis for two reasons:(1) first, they should be distinguished from ENMs or
contaminating NMs [27]; and,(2) second, due to their specific physico-chemical prop-
erties, NPs could interact with proteins, lipids, car-bohydrates, nucleic acids, ions, minerals andwater in food, feed and biological tissues.
It is important to characterize the effects and the inter-actions of ENMs in the relevant food matrix. Proteins andcarbohydrates have large specific surface areas and a highelectrochemical surface charge that is likely to make theminteract with charged particles, like many engineeringNPs (ENPs). These components also contain hydrophobicdomains that are likely to interact with hydrophobic ENPs[e.g., fullerenes and carbon nanotubes (CNTs)].
Food also contains natural colloids and dissolved ions.Dispersed colloids are particles in the ENP range (1–200 nm) that are kept in a stable aqueous suspension[11]. They do not precipitate by gravitation due to theirsmall size, a certain surface charge, electrostatic interac-tions, van der Waals forces and steric forces. Any changes(e.g., pH or ion concentrations) may destabilize the sus-pension. According to the classical double-layer and col-loid-stability theories, particle stability is affected by theconcentration of cations (coagulants), meaning that, atincreasing salt concentrations, free NPs will start toaggregate [20]. Releasing ENPs into such complex sys-tems is bound to lead to a range of interactions, and it is notevident whether a given ENP will be adsorbed to a surfaceor if it will be stabilized by natural polymers so that it re-mains mobile [37].
Given the huge diversity of ENPs for use in the foodand feed sector (e.g., chemical composition, size, sizedistribution, surface activity/modification) (see Table 1),and their potential interaction with food-matrix com-ponents (e.g., proteins), the determination of NMs in foodis a challenging task requiring tailored solutions. Asregulation for food nanotechnology moves forward, theaim of this review is to address and to compare theavailable analytical methods for determination of NMs infood. Two previous reviews underpin this one:(1) one provided a detailed description of food nano-
delivery systems and considered the analytical tech-niques useful for identifying and characterizingthese systems in food [38]; and,
(2) the other overviewed the different analytical tech-niques available for detecting the ENPs in product
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formulation, environmental matrices and food mate-rials [39], but it drew heavily on studies reportingcharacterization of NPs in raw products and environ-mental materials because limited work has been doneto date on detection and characterization of NPs infood.
This review takes a step forward by including the firstapplications of NMs to food.
2. Regulations
The safety of nanoproducts has attracted attention inline with their increasing use. Despite the rapid com-mercialization of nanotechnology, no specific nano reg-ulations exist anywhere in the world. Most regulatoryagencies remain in information-gathering mode, lackingthe legal and scientific tools, information and resourcesthat they need to oversee the exponential market growthof nanotechnology adequately [1,40–44].
At present, international organizations are stillattempting to determine the current capacity to assessthe health and safety risk associated with the use ofnanotechnology in food and food production and onsurfaces in contact with food.
Through its horizon-scanning activities, the Food andAgriculture Organization (FAO) has recognized the needfor scientific advice on any food-safety implications thatmay arise from the use of nanotechnologies in the foodand agriculture sectors.
With the FAO, the World Health Organization (WHO)has published the report of a Joint Expert Meeting held inJune 2009 on the topic of Application of Nanotechnolo-gies in the Food and Agriculture Sectors: Potential FoodSafety Implications [45]. This report presents an overviewof the wide range of current and projected nanotechnol-ogy applications in food and agriculture (Fig. 2). Appli-cations that may lead to human exposure to NPs throughthe environment to the food chain were not included.
The Council of the Organization for EconomicalCooperation and Development (OECD) has established aWorking Party on Manufactured NMs as a subsidiarybody of its Chemicals Committee [46]. This workingparty was established to address human health andenvironmental safety aspects of manufactured NMs inthe chemicals sector.
Furthermore, in February 2009, the European FoodSafety Authority (EFSA) published its opinion on the po-tential risks arising from nanoscience and nanotechnolo-gies in food and feed [47]. It considered, among otherthings, the suitability of current regulations relating to theuse of nanotechnologies in the food sector. The report didnot identify any major gaps in regulations, although itnoted that there was uncertainty in some areas as towhether applications of nanotechnologies would be picked
S d
P dNano-emulsionsSurfactant micelles
SpreadsMayonnaise
Procesednanostructured in food
Surfactant micellesEmulsion bilayers,
ayo a seCreamYoghurtsnanostructured in food
(NANOTEXTURES)y
Double or multiple emulsionsReverse micelles
Yoghurtsice creamsReverse micelles
N d li tNanomicelle-based carrier
Vegetable oil enriched in vitaminsNanodeliver systems
based on encapsulatedsystemNanocluster delivery system
Natural biopolymer from yeast cell walls that bind t i
Nanotechnology in Animal feedmycotoxinsPolystyrene (PS) base, polyethylene glycol (PEG) linker,
the agricultural sector Agrochemicalsy y ( ) , p y y g y ( ) ,
and mannose targeting biomolecule to bind E. coli Slow or controlled release fertilizers and pesticidesSlow- or controlled-release fertilizers and pesticides
Figure 2. Nanomaterials relevant to food.
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Table 2. European Union legislation applicable to NMs in food
Legislation Comments Ref.
General for chemical compoundsREACH European Community legislation concerned with chemicals and their safe use and dealing with the
Registration, Evaluation, Authorisation and restriction of CHemical substances.[51]
Novel foods regulationRegulation (EC) No 258/97 Novel foods are foods and food ingredients that have not been used for human consumption to a
significant degree in the EC before 15 May 1997, and the Regulation subjects all novel foods andfoods manufactured using novel processes to a mandatory pre-market approval system. In January2008, the European Commission published a proposal to revise and update the Novel FoodsRegulation. Various proposals have been discussed by the Commission, Parliament and Council (Thedraft Regulation is currently going through the co-decision procedure). A definition of NMs has beenintroduced at the request of the European Parliament, and supported by the Council. Discussions arecontinuing on how to bring nanotechnologies specifically into the revised Regulation.
Only additives explicitly authorized may be used in food. In December 2008, a new Regulation waspassed (Regulation EC/1333/2008), which set out a common authorization procedure for additives,enzymes and flavorings. From early 2010, a list of approved additives, including vitamins andminerals, came into force. Inclusion of additives on the list was decided by the Commission on thebasis of an Opinion from the EFSA. Those included often had limits set on their use, for example,restrictions on the quantities permitted for use. The new regulations also specify that, where thestarting material used, or the process by which an additive is produced, is significantly different (forexample, through a change in particle size), it must go through a fresh authorization process,including a new safety evaluation.
[54–56]
Food-contact materialsRegulation EC/1935/2004 All materials that are intended to come into contact with foodstuffs, either directly or indirectly. The
Commission or Member States may request the EFSA to conduct a safety evaluation of any substanceor compound used in the manufacture of a food contact material. Certain materials, includingplastics, are subject to additional measures. The Commission has proposed updating the Regulationgoverning food-contact plastics to specify that a deliberately-altered particle size should not be used,even behind a migration barrier, without specific authorization.
[57]
Food supplementsDirective 2002/46/EC States that only vitamins and minerals on an approved list may be used as food supplements. New
substances may be considered for inclusion on the list, but only after a safety assessment by EFSA.[58]
Trends Trends in Analytical Chemistry, Vol. 30, No. 1, 2011
up consistently. It concluded that ‘‘on the basis of currentinformation, most potential uses of nanotechnologies thatcould affect the food area would come under some form ofapproval process before being permitted for use’’.
These international agencies identified a number ofdomains of interest as a starting point for this review, sothat more research on NMs is needed to improve the basisof scientific knowledge in support of regulatory work:
– development of reliable measurement methods, ref-erence materials and materials characterization;
– review and development of test methods for humanhealth, safety and the environment;
– development of exposure information throughoutthe life-cycle of NMs;
– review of existing risk-assessment methods;– risk management for workers� protection;– networking existing and establishing new infra-
structures to examine health, safety and environ-mental aspects of NMs.
However, different countries are trying to include NMsin their current regulations. In USA, the EnvironmentalProtection Agency (EPA) is already empowered to
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regulate NMs under several laws. The EPA could usemost of the environmental laws – Clean Water Act(CWA), Clean Air Act (CAA), Comprehensive Environ-mental Response, Compensation and Liability Act(CERCLA), Resource Conservation and Recovery Act(RCRA), Federal Insecticide, Fungicide and RodenticideAct (FIFRA), and Toxic Substances Control Act (TSCA).
The US Food and Drug Administration (FDA) requiresmanufacturers to demonstrate that food and theiringredients are not dangerous to health, but there are nospecific rules for NPs because it regulates products, nottechnologies. Nevertheless, the FDA expects that manynanotechnology products will come under its jurisdic-tion. The FDA regulates a wide range of products,including foods, cosmetics, drugs, devices, and veteri-nary products, some of which contain NMs or are pro-duced nanotechnologically. The Acting Commissioner ofthe FDA initiated the Nanotechnology Task Force in2006 to help address questions regarding adequacy andapplication by regulatory authorities [48].
The European Commission (EC) aims to reinforcenanotechnology and, at the same time, enhance support
Table 3. Different parameters and characterization methods for NMs
Parameters Characterization methods Ref.
Particle size & size distribution,morphology or shape andaggregation state
Microscopy and microscopy-related (imaging): near field-scanning opticalmicroscopy (NSOM); scanning probe microscopy (SPM); confocal laserscanning microscopy (CLSM); scanning electron microscopy (SEM);transmission electron microscopy (TEM); scanning transmission electronmicroscopy (STEM); X-ray microscopy (XRM); scanning transmission X-raymicroscopy (STXM); and, atomic force microscopy (AFM)
[22,23,26,27,38,39,59–62]
Centrifugation and filtration [23,26,38,39,63]Chromatography and related (separation): size exclusion chromatography(SEC); capillary electrophoresis (CE); hydrodynamic chromatography(HDC); field flow fractionation (FFF)
mercury porositometry; and, laser diffractrometry [38,39,59]Chemical characterization Analytical (spectroscopy) coupled to electron microscopy (imaging): TEM-
dispersive X-ray spectroscopy (EDS); SEM-EDS; TEM-electron energy lossspectroscopy (EELS); TEM-selected area electron diffraction (SAED); and,AFM-chemical force microscopy (CFM)
[22,23,27,38,39,59–62]
Spectroscopy and related (characterization): Raman spectroscopy; LIF;UV-Vis; infrared spectroscopy; NMR
Chemical analysis of surface Spectroscopy and related (characterization): static X-ray spectroscopy [X-ray photoelectron (XPX); X-ray fluorescence (XRF); X-ray absorptionspectroscopy (XAS); and, X-ray diffraction (XRD)]
[22,38,39]
Mass spectrometry (characterization):Sources – electrospray ionization (ESI); matrix-assisted laser desorption/ionization (MALDI)); laser desorption/ionization (LDI); and, inductivelycoupled plasma (ICP)Mass analyzers – time-of-flight (TOF); quadrupole linear ion-trap (QqLIT);ion trap (IT); single quadrupole; triple quadrupole (QqQ); quadruple time-of-flight (QqTOF); static secondary ion mass spectrometry (SSIM)
[39,65–67]
Carrier-drug interaction Differential scanning calorimetry [39,68,69]Nanoparticle-dispersion stability Critical flocculation temperature(CFT) [39,70,71]Release profile In-vitro release characteristic under physiologic & sink condition [39]Target-compound stability Bioassay of target compound extracted from NP, chemical analysis of the
target compounds[39]
Trends in Analytical Chemistry, Vol. 30, No. 1, 2011 Trends
for collaborative research and development (R&D) on thepotential impact of nanotechnology on human healthand the environment via toxicological and ecotoxico-logical studies. The EC is performing a regulatoryinventory, covering EU regulatory frameworks that areapplicable to NMs (e.g., chemicals, worker protection,environmental, and product-specific). The purpose ofthis inventory is to examine and, where appropriate, topropose adaptations of EU regulations in relevant sectors[49]. This includes to apply existing food laws to foodproducts using nanotechnology. All food products have
to meet a general safety requirement under the GeneralPrinciples of Food Law Regulation (EC/178/2002) [50].More specific legislation covers the use of novel foods,food additives and food-contact materials (see Table 2).
3. Analytical approaches to characterize anddetermine NMs
Recently, the problem of NM safety, once mainly limitedto its chemical aspects, has been extended to possible
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toxicity associated with NPs as physical entities [22], sothe analysis of NMs commonly requires two types ofmethod:(1) those for characterizing and/or detecting NPs or
NMs; and,(2) those for determining their chemical composition.
However, the line that divides these types is very thin,taking into account that the chemical composition ofNMs is one of their properties. As outlined in Table 3,there are a number of analytical tools for qualitative andquantitative categorization of NMs:(1) single-particle techniques;(2) techniques characterizing the ensemble of NMs;
and,(3) techniques to determine their chemical composi-
tion.For a wide discussion on the advantages or the
drawbacks of each technique, we refer the reader toexcellent reviews by Tiede et al. [39] and Luykx et al.[38], who accurately explain their advantages and lim-itations, so there is no need for a repeat in this article.The main problem of analyzing NMs in food is that mostof these analytical systems have been used to charac-terize the NM themselves and only few are applicable tothe analysis of more complex samples. Food is a veryintricate material, and, probably, for sufficient charac-terization, the NMs need to be separated from the foodmatrix, as Luykx et al. [38] discussed for nanodeliverysystems. However, the tendency is to reduce samplepreparation as much as possible because it is importantto measure the NMs in the relevant matrix, as theirproperties may depend on the surrounding matrix andbe affected by processing as well as by the extractionprocedure. This is usually much more demanding thanto analyze NMs in simpler or model matrices.
With nanoscale metals or semiconductors containingNMs, these can be detected even in rather complexmatrices (e.g., food, feed and biological tissues) by meansof electron microscopy (EM) coupled with chemicalanalytical tools. However, detection by EM is only pos-sible if the number of NMs is sufficiently high in thematrix to localize them, since high magnification is re-quired due the small size of NMs. As a result, theinvestigation of NM distribution in food is generally ex-tremely time consuming. We need to mention thattransmission EM (TEM) has so far provided the mostdetailed information regarding NM location by allowingboth visualization of the location within food, and, inconjunction with spectroscopic methods, characteriza-tion of the composition of the internalized NPs.
Visualization is most easily accomplished with elec-tron-dense NMs (e.g., metal NPs). However, the tech-nique is also suitable for any other NMs, includingorganic compounds (e.g., fullerenes and CNTs). How-ever, the long time required for both sample preparationand image analysis greatly limits the analytical
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throughput when using these techniques for analysis ofNPs in food.
For CNTs, Raman spectroscopy is a very importanttechnique and could be very useful for finding CNTs andfullerenes. This technique is cheap, non-destructive andnot time consuming. It has also the advantage of beingused in in situ monitoring. Several reviews highlight thepower of Raman spectroscopy for measuring CNTs[72,73].
The second group of techniques possesses a loweranalytical ambition but is best suited to routine analysis.These approaches are based on the chemical character-ization of NMs, without generating information on theirphysical state. Hence, the metal content of NMs can bequantified by analytical-chemistry tools [e.g., induc-tively-coupled plasma atomic emission spectroscopy(ICP-AES) or mass spectrometry (ICP-MS)], or by radio-analysis after appropriate neutron irradiation. Generally,sample preparation includes acidic sample digestionbefore analysis. The limitations of chemical analysisresult from artificial losses during the preparatory steps,analytical detection limits and the inability to charac-terize carbon NPs (e.g., polymeric NPs, fullerenes, andCNTs). In the case of organic NMs, detection or quanti-fication of the chemical may be possible, where a test forthe species exists, but a focus on characteristic structuresmay be needed to determine whether it is still in nano-form.
So far, only very limited work has been done on thedetection of organic NPs in food. However, the need todetermine organic NMs and nano-delivery systems ledscientists to imagine what techniques could also beapplicable to their characterization in food, even thoughthey have not yet been used. There are a few recentexamples of NP-enabled MS that, though not explicitlylinked to NP determination in food, demonstrated thepotential of this approach. For example, matrix-assistedlaser desorption/ionization (MALDI)-time-of-flight (TOF)-MS and laser desorption/ionization (LDI)-TOF-MS wereuseful for characterization of ultrasmall NPs of TiO2
[67]. The size distributions of TiO2 NPs obtained fromMALDI-TOF-MS and LDI-TOF-MS were in good agree-ment with parallel TEM observations.
TOF secondary-ion MS (SIMS), fluorescence micros-copy and scanning EM (SEM) were employed to monitorthe immobilization of biotinylated shell-crosslinked NPson biotin/streptavidin-functionalized, UV-photo pat-terned self-assembled monolayers [74].
There are also some studies reporting quantification ofNMs in a related field (e.g., environmental analysis),where much more information is available. Isaacson andBouchard [75] reported the first methods for the asym-metric flow field-flow fractionation (AF4) size separationof aqueous C60 aggregates in deionized water withoutuse of mobile-phase modifiers coupled with in-line dy-namic light scattering (DLS) and off-line with liquid
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chromatography with atmospheric pressure photo-ioni-zation MS (LC-APPI-MS).
Meanwhile, Farre et al. [76] gave details of the firstdetermination of C60 and C70 fullerenes and N-methyl-fulleropyrrolidine C60 on the suspended material ofwastewater effluents by LC hybrid quadrupole linear iontrap tandem MS. As we go deeper into the analysis ofNPs, we need analytical procedures to account for ex-plicit information, including molecular weight and thenumber and the identity of functional groups. Sensitiveand mass-selective detection, as offered for MS combinedwith optimal extraction procedures, shows great poten-tial to achieve this goal.
There are some additional complications not yetsolved in the analysis of NMs, such as the fact that someENMs cannot be distinguished from naturally-occurringvariants of the same {e.g., nanoscale engineered silicondioxide (SiO2) or endogenous lipids used in capsulemembranes}. Detection may also be hindered by inter-actions with solutes or food components that obscureclear analytical signals. Despite some successes in thisfield, its evolution seems to be hampered by the limitedinformation available. This will be a growing area withinNM analysis.
Moreover, there remain several obstacles to obtainingadequate characterization and quantification of NMs infood. Foremost among them are those presented by thelack of analytical standards, relevant reference materialsand internationally standardized practices, protocols, andprocedures for testing the preparation of food, NPmeasurement, and data analysis. The OECD has estab-lished a list of prioritized materials that takes into accountthose materials that are already in production (or close tocommercial use), as well as considerations of productionvolume, the likely availability of materials for testing andthe existing information [46]. The OECD list comprises:� fullerenes (e.g., C60);� single-walled and multi-walled CNTs (SWCNTs
and MWCNTs, respectively);� carbon black;� polystyrene;� dendrimers;� nanoclays; and,� NPs of Ag, Fe, TiO2, Al2O3, CeO2, ZnO, and SiO2
[46].Internal standard quantification using stable isotope-
labeled internal standard is de rigueur for trace-levelanalysis of contaminants in food. Currently, there iscommercially available only a single, stable-isotope la-beled fullerene internal standard, 13C60. The currentlimited number of standardized reference materials forengineered NMs is another brake on precise, reproduc-ible detection and quantification of engineered NMs infood and feed.
The Joint Research Centre, Institute of ReferenceMaterials and Measurements, has recently released a
quality-control material (IRMM-304) of silica NPs. Thereare also available gold NPs (NIST RM 8011, 8012 and8013) and polystyrene spheres (NIST SRM 1963a and1964) from the National Institute of Standards andTechnology (NIST) [77].
Furthermore, interlaboratory studies, which are nec-essary for validation of protocols and generating preci-sion and bias statements for measurement standards, arealmost non-existent.
Of the difficulties encountered in conducting inter-laboratory studies on these emerging contaminants, themajor problem is the wide range of analytical techniquesapplicable to these compounds and the different purposesof the methods developed [78,79]. There is much workyet to be done to show how far determination of NMs infood can go.
4. Applications to food analysis
Applications of the analytical methodology reported inthe previous section to the analysis of NMs in food arestill very scarce. Table 4 lists the results of the literaturesearch. Various approaches suggested for use in studiesof NP bioaccumulation are included because they can beapplied to its determination in food, since many of thetarget organisms are also edible.
We need to mention that methods to determine inor-ganic NPs in food, especially aquatic organisms, arequite developed. There are two main types of procedure:(1) those combining microscopy and spectroscopy to
identify and to characterize the NMs as well as todetect their chemical composition; and,
(2) those looking at their chemical composition only.For inorganic NPs, the latter methods are based
mainly on wet digestion with a strong acid (e.g., nitric orperchloric) followed by ICP optical emission spectroscopy(ICP-OES) or ICP-MS. For example, several methods tomeasure gold NPs have been developed. These methodsare based only on chemical analysis, since we may as-sume that all the gold present in the samples is from NPs,since gold is not abundant in the environment. Con-trarily, other metal NMs {e.g., unmodified commercialnanoscale metal oxides, zinc oxide (ZnO), cerium dioxide(CeO2) and titanium dioxide (TiO2)} were characterizedby TEM and environmental scanning EM (ESEM) withenergy dispersive X-ray analysis (EDX) elemental anal-ysis to establish their structure as NPs [81].
Also, fish samples were digested with acid and ana-lyzed by ICP-MS. Definitive uptake from the watercolumn and location of TiO2 NPs in gills was demon-strated for the first time by using coherent anti-StokesRaman scattering (CARS) microscopy. CARS imaging ofrainbow-trout gill tissues clearly showed large aggre-gates of TiO2 (up to 3 lm) on the surface of the gillepithelium following 24–96 h exposure (Fig. 3).
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Table 4. Selected applications for detection, characterization and/or quantification of NMs in food or food like matrices
Problem Nanomaterial Matrix Techniques Comment Ref.
Food and beveragesInvestigation of thepresence of microsizedand nanosizedcontaminants
InorganicNMs
Bread andbiscuits
ESEM/EDS Detection of organic andinorganic microscale andnanoscale contaminants: ESEMIdentification of their chemicalcomposition: EDS
[80]
Bioavailability ofnanoscale metal oxidesto fish
TiO2, CeO2
and ZnORainbow trout TEM
ESEM/EDSICP-MSICP-OESCARS
Characterization of size, particleshape/morphology andqualitative aggregation: TEM andESEM/EDSAnalysis of elements content :wet digestion and ICP-MS, ICP-OESConfirmation of the presence ofthe NP in fish: CARS
[81]
Bioaccumulation of goldNPs in fish
Au NPs Mytilus edulis ICP-OES Analysis of Au content: wetdigestion with nitric acid andhydrogen peroxide and ICP-OES
[82,83]
In vivo toxicity studies FullerenesC70�C98
Embryo zebrafish
LC-MS2 Digestion with glacial acetic acidand toluene150 mm · 2 mm Targa C18
column and toluene/methanol(55:45) isocratic mobile phaseOnly molecular ions wereobtained
TPEM combined withautofluorescence can be used todetect NMs interacting withvegetation and TPEM can beused simultaneously to detectand to monitor the interactions ofMWCNTs and PAHs in vivo inroots.
[85]
Migration and stability studies with food-packaging materialsDetect clay NPs andcharacterize their size
Biopolymer polylactidewith 5% Cloisite30B as filler
95% ethanol asfood simulant
XRDTEMCentrifugationFA-MALSICP-MSFA4-MALS-ICP-MS
Characterization of polylactide/Cloisite 30 B: XRD, TEMChemical characterization ofelements: ICP-MSSeparation and sizedetermination of clays:centrifugation, FA4-MALS andclay aspect ratioMigration study FA-MALS-ICP-MS
[86]
Stability of NPs duringheat treatment
Chitosan NPs forl-ascorbic acid
Aqueoussolutions
Zeta potential PDIUltracentrifugation
Characteristics of AA-loaded CSnanoparticles: particle size, zetapotential, encapsulationefficiency (EE), and releaseeffectsStability of AA-loaded CSnanoparticles with the changes ofphysico-chemical properties andrelease rate before and after heatprocessing in aqueous solutionsat various temperatures.
[87]
CARS, Coherent anti-Stokes Raman scattering microscopy; ESEM/EDS, Environmental scanning electron microscopy/X-ray microprobe of anenergy dispersive system; FA4, Asymmetrical flow field-flow fractionation; ICP-MS, Inductively coupled plasma mass spectrometry; ICP-OES,Inductively coupled plasma optical emission spectroscopy; LC-MS2, Liquid chromatography-tandem mass spectroscopy; MALS, Multi-anglelight-scattering detection; PDI, Polydispersity index; TEM, Transmission electron microscopy; TPEM, Two-photon excitation microscopy, XRD,X-ray diffraction.
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Figure 3. Coherent anti-Stokes Raman scattering (CARS) microscopy images of the gill tissue of rainbow trout, Oncorhynchus mykiss, followingwater-borne exposure to titanium dioxide (TiO2) nanoparticles (NPs). The cellular structure of the primary (PL) and secondary (SL) gill lamellae,composed of pillar cells (PCs) and pavement cells (PVs), was obtained by epidetection of the CH2 vibration (shown in green). The red blood cellsare effectively separated from the lamellae cells by forward detection of the CH2 vibration (shown in blue). (A) Gill tissue, following a 28-dayexposure. An aggregate of NPs can be seen occupying the space between the pillar cells. (B) The same NP aggregate under a 3· increase inmagnification. (C) Projection of a 300 · 100 lm 3D data set of gill tissue following a 14-day exposure. A cluster of NPs can be seen in the regionof the marginal channel (MC). (D) Multi-planar view of the same exposure. The two adjacent sub-panels specifically locate the NPs inside thetissue near the surface of the MC. (Reproduced from [81] with permission, ª 2010 American Chemical Society).
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Gatti et al. [80] investigated the presence of inorganicmicro-sized and nano-sized contaminants in bread andbiscuits from 14 different countries by ESEM. EDX wasemployed to identify their chemical composition. Theresults indicated that 40% of the samples analyzedcontained foreign bodies (e.g., ceramic and metallicdebris, probably of environmental or industrial origin).Fig. 4 shows other debris found in a biscuit, whichcontained cadmium, silver, tungsten, aluminum, sulfur,calcium, iron, cobalt and copper in particles.
Biopolymer nanocomposites are a field of emerginginterest, since such materials can exhibit improvedmechanical and barrier properties, and they can be moresuitable for a wider range of food-packaging applications.
From a food-safety point of view, it is important tocharacterize migrates from nanocomposites containingclay as fillers. Schmidt et al. [86] demonstrated that AF4
with multi-angle light scattering (MALS) detection wasuseful for characterizing the size of NPs contained inmigrates from nanocomposites of polylactide andorganomodified montmorillonite clay as filler. However,this coupled instrumentation alone did not provide anyinformation on the identity of the NPs occurring in thesimulated food. This limitation was overcome by cou-pling AF4-MALS to the element-selective ICP-MS detec-tor, which provided additional information on traceelements known to be naturally present in the clay. Theanalytical system was applied to characterize migrates
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Figure 4. Environmental scanning electron microscopy (ESEM) image (A) of debris found in a sample of bread from South Italy made with hardwheat with its energy-dispersive system (EDS) spectrum and the semi-quantitative concentrations of the elements (B). It contains micro-scale andnano-scale cadmium-tungsten-silver-cobalt contamination. (Reproduced from [80] with permission, ª 2009 Taylor and Francis Group).
Figure 5. Selected chromatograms after liquid chromatography with electrospray ionization mass spectrometry (LC/ESI-MS) with methanol/tol-uene (80:20) unless otherwise noted, including C60 (2 lg/L in zebrafish homogenate matrix), 13C60 (10 lg/L in zebrafish homogenate matrix), C70
(10 lg/L), C82 (3.4 lg/L), C88 (2.5 lg/L), and C98 (0.4 lg/L). Additional fullerenes in a higher-order mixture not shown. (Reproduced from [84]with permission, ª 2007 American Chemical Society).
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from nanocomposite films of polylactide and theorganomodified Cloisite 30B montmorillonite clay usedas filler. The results demonstrated that NPs of 50–800 nm radius indeed migrated from the nanocompos-ite, but ICP-MS signals corresponding to clay mineralswere absent.
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Isaacson et al. [84] developed and validated an ana-lytical method to quantify a suite of fullerenes and thenapply the analytical method to determine the behavior ofa single fullerene, C60, during a toxicological assay usingzebrafish embryos and aqueous-exposure solutions. Theaverage recovery of C60 from fish extracts was 90% and
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precision, as indicated by the relative standard deviation,was 2%. The method quantification limit was 0.40 lg/kg.LC-ESI-MS detection was used to identify and to quantifyC60–C98 fullerenes. The most abundant ions formed underESI-MS conditions were molecular ions. The mobile phase(55:45 toluene/methanol) provided chromatographicseparation of the fullerene analytes using a conventionalC18 analytical column. Chromatographic analysis indi-cated co-elution of C60 with the 13C60 internal standardwith the retention times of the larger fullerenes increasingin order of increasing carbon number (Fig. 5).
Two-photon excitation microscopy (TPEM) combinedwith autofluorescence was used to detect NMs(MWCNTs, TiO2, and CeO2) interacting with vegetationand used simultaneously to detect and to monitor theinteractions of MWCNTs and polycyclic aromatichydrocarbons in vivo in roots [85]. The potential ofTPEM coupled with autofluorescence in visualizingMWCNTs and their interactions with in vivo cellularsystems is both extensive and diverse, and highlights thetechnique�s potential for use with other NMs. It may alsoprovide a method for looking at the purity of NM man-ufacture, where artifacts (e.g., catalysts from the man-ufacturing process) can be identified rapidly from theirautofluorescence signals. TPEM combined with auto-fluorescence provides a non-intrusive tool for the in vivovisualization of NM fate, interactions and behavior.Future applications may include studies on NM envi-ronmental fate, bioavailability, ecotoxicology, chemicalcarriage, and targeted drug delivery in systems fromplants and bacteria to skin or synthetics. Experience withthe combination of TPEM and autofluorescence is veryscarce, but, again, the results are promising.
5. Conclusions and future trends
Analysis of NMs in food, as has been widely remarkedupon in the literature, is still in its earliest infancy, eventhough there are methods that have proved their effec-tiveness in detecting and characterizing NMs. However,the methods to quantify them are still rare.
At present, the situation is divided. On the one hand,there are methods to analyze the structural form of NMsin food, feed and biological tissues, but, because of thebackground occurrence of NMs, it is not usually possibleto establish the presence of ENMs. On the other hand,there are methods to analyze the chemicals in specificENMs, but most often not to establish their presence innanoform. At present, only in exceptional cases is itpossible both to detect specifically and to measure par-ticular ENMs, and, in these cases, that is feasible thanksto the combination of a number of analytical techniques.
However, if the results compiled in this review arecompared with the data presented in other previoustreatments of this topic, we can highlight the rapid
progress that represents a major step forward and laysthe foundation to develop quantitative methods for theanalysis of NM residues in foods. There are a growingnumber of these quantitative analytical methods, and weexpect impressive applications within food safety, foodquality and food analysis in the very near future, as anextension of those already existing for environmentalsamples. An important driver for their development isthe concern for the food safety of international agenciesand organizations (FAO/WHO, EU, EFSA, and US EPA)arising from the possible toxic effects of NMs. Theassessment of the risk to consumers needs to be ad-dressed as regulations for food nanotechnology moveforward.
Summarizing, due to the enormous variety of NMs,there are many different ways to analyze particles andthere is no best technique for all situations, so a com-bination of techniques is usually necessary.
AcknowledgmentsThe authors thank the Spanish Ministry of Educationand Science and the European Regional DevelopmentFunds (ERDF) (Project CGL2007-66687-C02-01/BOS)for financial support.
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