The Presence of Nanoparticles in Food Product and the Challenge in Sample Preparation and Detection WANG Zheng Ming AVA Spectroscopy Solutions 2014 eConference May 2014 Presentation Summary -General nanotechnology: development and application -Nanotechnology and agro/food products -Presence of nanoparticles (NPs) in food products -Human exposure of ENPs through food products -Analytical methods and techniques -Examples: analysing ENPs in complex matrix -Conclusion Nanotechnology – Development and Applications Yesterday Today & Tomorrow Passive Nanostructures ● Nanoparticle ● Polymers ● Nanocoatings ● Nanostructured metals 1. Gen 2000 2. Gen Nanosystems Active Nanostructures ● Guided assembling ● Amplifiers ● Sensors ● Targeted drugs ● Adaptive structures ● 3D Networks & new hierarchical architectures ● Robotics Molecular Nanosystems ● Molecule device by design ● Atomic design; ● Emerging functions 4. Gen Converging Technologies ● Nano-bio-info from nanoscale ● Cognitive technologies; ● Large complex systems (nanoscale) 3. Gen 2010 today 2020 Year Adopted from MC Roco. AIChEJ 50:890 (2004) Advanced Nanotechnology & Consumer Products Today MC Roco (2011) J Nanopart Res 13:427–445 Generally, nanotechnology deals with structures sized between 1 and 100 nm in at least one dimension. Guidance on the risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain. EFSA Journal 2011;9(5):2140 On scale, this tennis ball is the same size in relation to earth as a nanoparticle is to a tennis ball. Head of pin 1 millimetre = 1,000,000 nanometers Red Blood Cell 2.5 micrometers Ragweed pollen 20 micrometers Carbon nanotube 2 nanometers = 2,500 nanometers = 20,000 nanometers = 2 nanometers Info from: http://nanohex.org/flash/Nano_What.html Engineered Nanoparticles (ENPs). Different forms substances, shapes, & sizes. Particles, / Rods, Fibres and Tubes / Sheets / Composites / Aggregates, Agglomerates, Encapsulates, etc… Unique physicochemical properties: -high surface-to-mass ratio and surface reactivity; -they are expected to interact with substances such as proteins, lipids, carbohydrates, nucleic acids, ions, minerals and water present in food and biological tissues; Some ENPs are designed to cross the blood-brain barrier PNAS (2011) 108(46):18837-42 J Control Release. (2012) 161(2):264-73 J Drug Target. (2004) 12(9-10):635-41 Nanotech & medicine Consumer Products Using Nanotech Applications -Cosmetics and personal care products; -Paint and Coating; -Catalysis & Lubricants; - Security printing; -Textiles and sports; -Food and nutritional supplements; -Food packaging; -Agrochemical; -Construction materials; - Fuel Cell & batteries; -Paper manufacturing; -Water decontamination; -Others Woodrow Wilson Databank, http://www.nanotechproject.org/cpi/ Nanotechnology and Agro/food products Food production must increase 70-100% (FAO 2009 estimation) Food Security Agro Technology Food Science and Technology World Population Growth Population (billions) -world population -meat consumption -available crop land -water tables -climate change 9.3 billion 6.9 billion Year Source: UN, Department of Economic and Social Affairs, Population Division(2011) Nanotechnology Application of Nanotechnology in Agriculture Sectors Nanosystem Precision farming maximizing crop yields (output), minimizing input (i.e. pesticides, fertilizers, and herbicides, etc) through efficient monitoring environmental variables and applying targeted action, also improve soil sustainability System Component: 1) remote smart / nano sensors for real time crop development, soil condition, usage of agro-chemicals, seeding, etc… 2) GPS; 3) IT system and 4) controlled release Nanoforum Report (2006): Nanotechnology in Agriculture and Food Application of Nanotechnology In Food Products Application Nano-textured food ingredients Nano-delivery systems for nutrients / supplements Organic & Inorganic nano-sized additives for food -Surface functionalized nano materials -Nano-coatings on food contact surfaces Nanotechnology Function Processed nano-structures Novel or improved tastes, flavours, in food textures Nano-encapsulated Nanocarrier systems used for taste bioactive substances – masking of ingredients / additives mainly additives and i.e. fish oils, protection from supplements degradation. manufactured in the Due to larger surface area, smaller nanosize range quantity would be needed. -2nd gen ENPs add functionality to the matrix, i.e. antimicrobial activity via O2 absorption. -Nanoscale coating. -functionalized ENPs to bind the polymer matrix, provide a barrier against volatile flavours or moisture movement, - Nanocoatings for FCMs with barrier or antimicrobial properties. modified from 1) FAO/WHO (Nov. 2012) State of the art on the initiatives and activities relevant to risk assessment and risk management of nanotechnologies in the food and agriculture sectors 2) FAO/WHO (2010). FAO/WHO expert meeting on the application of nanotechnologies in the Food and agriculture sectors. Potential food safety implications. Meeting report. Food Products Examples Ag (FCM) NP Ag and NP TiO2 will be used as example for sample prep and detection later TiO2 (anticaking agent) Woodrow Wilson Databank http://www.nanotechproject.org/ Food and food related products incorporating nanotechnology include nanoscale fertilizers, pesticides, food additives, enzymes, flavourings, and food packaging materials. Farm Primary Production Supply Seeds Fertilizers Feed, etc Distribution Network Processing /Packaging Consumers Retail Stores Nanotechnology has an impact on every aspect of the food supply chain, from how food is cultivated, produced, processed, packaged and stored. Emerging technologies governance is essential. Human potential and technological development are coevolving with benefits and risks. Mike Roco, National Science Foundation and National Nanotechnology Initiative Nanomanufacturing Summit 2011, Boston, September 27, 2011 Sustainable Development is the KEY Presence of NPs in Agriculture / Food Products and Possible Routes of Human Exposure Water, soil, other contact skin and membranes Air respiratory system Food Food & Water Food and FCMs digestive system Not only direct food ingestion Residues from agriculture and industral production can enter into air, water, soil, etc… Fertilizer & Pesticide Plants Feed and Vet Drugs Animal "All things are poison, and nothing is without poison; only the dose permits something not to be poisonous.” Paracelsus (1493-1541) [pærəˈsɛlsəs] Examples: Salt & sugar How much is too much? Nano particles ??? Father of the toxicology The need for characterization & quantification risk = hazard x exposure Risk Assessment Paradigm is Applicable Fadeel & Garcia-Bennett (2010) Adv Drug Del Rev 62:362 Challenges complexity dynamics trans-disciplinarity uncertainty Challenges Food Product Application Specific Both are interconnected Challenges Nanotech Development and its Application in General Challenges ENPs in Food Matrix -dynamic interaction of ENPs with food matrix constituents -aggregation due to food matrix environment, -broad variety of matrices (raw and processed food) (influence their sample preparation, detection, and risk assessment) Risk assessment Characterization of ENPs in five stages: (1) Pristine state (as manufactured); (2) As delivered to be used in food/feed; (3) As present in food/feed matrix; (4) As present in biological matrices; (5) As toxicological tested; Do we have the comprehensive knowledge? -Limited practical RA experiences in the food/feed areas -RA on a case by case basis (EFSA) -Data for long term / low dose exposure missing -Info on shorter term exposure not reliable -International harmonization needs Complexity in exposure consideration Challenges -Globalized food supply chains: Ingredient of food products can come from different regions (cottonseed oil and olive oil) -Exposure to food and water with incidental nanomaterials may have high risk -TiO2 – E171 Not specifically labelled as nanosized. However,….…. -Bioaccumulating and persistent NPs likely end up in the food / feed chain as contaminants -Nano applications and organic food Very little dietary information, especially toxicokinetic information, is available for Nanoparticles -- Uncertainty Legislation & Labelling : Synchronized and Asynchronous Environ. Sci. Technol. 2012, 46, 2242 Challenges The development of analytical techniques is a key to understand the benefits as well as the risks of the application Of nano materials in food products Characterisation Parameters of ENPs used in food products Size and size distribution NPs defined and classed by their size, the primary properties describing transport behaviour Morphology and shape It affects NPs’ surface areas and surface-to-mass ratio, it also can posses different affinities or accessibilities Elemental composition different particle composition  different behaviour, toxicity, impact Mass concentration normally it correlates its toxicity/impact, but this is not always applicable for NPs; Particle number NPs have low mass concentrations, but show high percentage of total particle numbers Aggregation state NPs have a tendency to aggregate, increase in size could lead to decrease in uptake Surface area (e.g. porosity) Surface charge Surface chemistry Increase in surface area, reactivity and absorption behaviour influence on particle stability especially in dispersions Coatings can consist of different chemical compositions and influence particle behaviour or toxicity Solubility soluble NPs, their ionic form can be harmful or toxic Structure The structure can influence stability or behaviour Modified from Tiede at al. 2008. Food Additives and Contaminants 25(7) 795–821 Nanoparticles, aggregates and agglomerates primary particle (pristine) agglomerated primary particles aggregated primary particles Different sample preparation for sample contains NPs’ aggregates and agglomerates will affect the detection outcome. Adopted from Peters and Bouwmeester of RIKILT Analytical methods for NPs characterization Microscopy related techniques STEM, TEM, SEM : AEM, CFM: size, size distribution, Morphology /shape, aggregation, structure, SEM/TEM mass concentration, surface chemistry Field-Flow Fractionation Separation / fractionation Chromatography related techniques HDC, FFF: ICP-MS: size distribution elemental analysis (quantitative) ICP-MS Elemental analysis / quantification (metal / metaloxide NPs) Modified from Tiede at al. 2008. Food Additives and Contaminants 25(7) 795–821 Analytical methods for NPs characterization Spectroscopic related techniques NMR: XRD: chemical composition aggregation, chemical composition, elemental analysis, structure, Centrifugation & filtration techniques UC, CFF: size distribution Other techniques Zeta potential: BET: surface charge, aggregation surface area & porosity Modified from Tiede at al. 2008. Food Additives and Contaminants 25(7) 795–821 Abbreviation physicochemical characterisation of ENPs will need different analytical methods STEM: scanning transmission electron microscopy TEM: transmission electron microscopy SEM: scanning electron microscopy AEM: analytical electron microscopy CFM: chemical force microscopy HDC: hydrodynamic chromatography FFF: field-flow fractionation ICP-MS: Inductively coupled plasma- Mass spectrometry NMR: Nuclear magnetic resonance XRD: X-ray diffraction UC: Ultracentrifugation CFF: Cross flow filtration BET: runauer–Emmett–Teller Common Analytical Approach Food safety tests -Presence -Identity chemical composition & size distribution -Concentration mass & particle number Screening: e,g, Image technique / in matrix, better suit for heavy elements, special sample prep, automated imaging analysis Confirmatory methods: e.g. FFF on-line coupling ICP-MS quantification unambiguous identification [sample prep: extract NPs from sample matrix, removal interfering matrix component enrichment of targeted analytes] Examples 1 A method is developed and validated for sizing and quantifying NP Ag in chicken meat using SP ICP-MS Development and validation of single particle ICP-MS for sizing and quantitative determination of nano-silver in chicken meat Anal Bioanal Chem. 2014 Jan 5. [Epub ahead of print] Ruud J. B. Peters & Zahira Herrera Rivera & Greet van Bemmel & Hans J. P. Marvin & Stefan Weigel & Hans Bouwmeester RIKILT, Wageningen University Research, The Netherlands Sample prep -chicken meat purchased from local supermarket. -200mg subsample, cut into small pieces, placed into a 10mL PE tube. -the subsample in the tube was fortified with a 50mg/L aqueous suspension of the 60nm Ag NPs at 5, 10, and 25 mg/kg. -two steps enzymatic digestion -1st, add 4 mL of the digestion buffer, vortex vigorously 1 min tip sonication at 4W power 5 min / tube on ice. -2nd, add 25μL of proteinase K, incubation for 3 h at 35 °C. -cooling to room temperature, -dilute the digest 100,000 times and measured using sp-ICP-MS. Anal Bioanal Chem. 2014 Jan 5. [Epub ahead of print] Method validation: repeatability, reproducibility, trueness, linearity (see below picture), LOD/LOQ, robustness, specificity/selectivity. Linearity demonstrated by the analyses of matrix-matched standards of 60-nm Ag NPs in the range of 0.05Vl–5VL on each of the validation days Anal Bioanal Chem. 2014 Jan 5. [Epub ahead of print] EM To determine the fate of NP Ag after addition to the chicken meat. 0 hr: the digest of sample processed directly after adding NP Ag 48 hr: the digest was produced 48 h after adding the NP Ag to the sample. Anal Bioanal Chem. 2014 Jan 5. [Epub ahead of print] EM pictures and EDX spectra of chicken digests The EDX spectra show that pure silver is partly transformed into silver sulfide Anal Bioanal Chem. 2014 Jan 5. [Epub ahead of print] Characterisation of TiO2 NPs in sunscreens using FFF on-line coupled ICP-MS Heidi Goenaga-Infante’s group @ LGC Limited J. Anal. At. Spectrom., 2012, 27, 1084 Examples 2 Comparison of extraction procedures for TiO2 NPs from sunscreen A. Contado & Pagnoni (2008) Anal. Chem. 80, 7594 B. Samontha et al. (2011) Anal. Bioanal. Chem. 399, 973 Sub-sample size 0.17–0.52 g 0.01 g First solvent 20 mL water 1 mL hexane (defatting first) Second solvent 20 mL methanol 1 mL water Third solvent 10 mL hexane (defatting later) — Defatting time 1 h phase separation 12 h, room temperature Separation of hexane Separation funnel Not reported Sonication Tip sonication, twice 15–60 s None Concentration of extract Rotary evaporation to remove methanol None Remarks: - More than 1 containers used -Using separation funnel -Loss of NPs to the tube walls -Only 1 container used -No separation funnel needed from J. Anal. At. Spectrom., 2012, 27, 1084 Defatting b/f NPs suspension reducing matrix interferences on the target particles A. Contado & Pagnoni (2008) Anal. Chem. 80, 7594 B. Samontha et al. (2011) Anal. Bioanal. Chem. 399, 973 from J. Anal. At. Spectrom., 2012, 27, 1084 Total Ti concentration and extraction efficiency were determined by ICP-MS Sample Solvent Ti [mg kg1] replicate 1 Ti [mg kg1] replicate 2 Extraction efficiency [%] (standard deviation) SPF 15 Water SPF 30 Water SPF 50 Water SPF 15 2.5% (v/v) hexane SPF 30 2.5% (v/v) hexane SPF 50 2.5% (v/v) hexane 36.3+ 1.8 66.0 +1.8 113.8 +1.7 41.6 +1.8 72.2 +1.8 195.8 +1.8 34.7 +1.7 54.3 +1.8 138.3 +1.8 33.6 +1.8 60.5 +1.8 195.2 +1.8 64.0 (0.5) 79 (9) 71 (13) 68 (8) 87 (7) 110 (5) NPs TiO2 loss On tube wall a The weight of sunscreen was in the range of 0.1003 g to 0.1061 g for all replicates. from J. Anal. At. Spectrom., 2012, 27, 1084 -Total Ti concentrations were determined by ICP-MS after microwave assisted digestion: 200 mg of sunscreen samples digested with a mixture of 2.5 mL nitric acid, 2.5 mL hydrogen peroxide and 0.5 mL hydrofluoric acid using a Multiwave 2000 microwave system with Teflon vessels. -Three TiO2 reference material (NIST 154c) subsamples were digested as described above. -The digests were made up to a total mass of 50 g with deionised water. Conclusion challenges remain in NPs analytical methods for food matrix -Diversity of NP types, combined with variety of food types / matrices -Interaction of the NPs with food matrices, behaviour unknown -NPs aggregation, agglomeration, affinity for surfaces – dynamic -NPs stability, in food sample and after extraction -Natural NPs present in food Conclusion challenges remain in NPs analytical methods for food matrix -Methods available for pure NPs, only few for complex matrices -Validation of methods , identify and quantify NPs in food matrix -Availability of Certified reference materials-well characterized and stable -Sample preparation and recovery of NPs, uncertainty, lack of workable standards / methods -Necessary to have different sample preparation protocols for different NPs added within different matrix In real situation, usually don’t know the type of NPs present in a food sample -Integrated and harmonized (internationally) analytical approaches Acknowledgement RIKILT: Ruud Peters & his colleagues LGC: Heidi Goenaga-Infante & her colleagues NTU: Kee Woei NG NUS: David LEONG AVA: CH’NG Ai Lee Tze Hoong CHUA Paul CHIEW CHEW Siang Thai Thanks