Electron microscopy as an analytical tool for detection and characterization of inorganic nanoparticles in food EU Framework Programme 7-Nanolyse
Nanostructures in food 1. Natural e.g. milk proteins 2. Unintentionally added: e.g. fumed silica- (S. Dekkers et al.,2010), contamination during processing e.g. (A. M. Gatti et al.,2009) 3. Food additives in Nanofoods
Nanotechnology in food industry 1. Food additives: Improved taste and texture Nanoencapsulation of nutrients Food preservation 2. Food packaging: Prolonged shelf life Intelligent packaging http://www.nanotechproject.org
Potential hazards of nanotechnology Different properties than the bulk analogue Ecotoxicity of nanomaterials has been reported Not enough data on fate of nanomaterials in biological systems and food products Toxicity to humans via ingestion? (I. Lynch et.al., 2009)
Nanotechnology in food applications- a challenge Challenge for regulation authorities Public opinion Detection and characterisation of nanomaterials in food
Why electron microscopy? Analytical tool in food structure analysis Used to characterize natural food nanostructures and engineered nanoparticles in environmental samples Can provide data on size, shape, distribution, agglomeration, chemical composition, presence and thickness of associated substances etc.
Drawbacks of using electron microscopy for food samples containing nanomaterials Sample preparation- alteration of the sample, loss of components Liquid and environmental electron microscopy- insufficient resolution? Distinguishing between engineered and natural nanostructures Is the sample representative? Cost Time consuming
Objectives Development of suitable sample preparation methods for imaging and screening of inorganic nanomaterials in food samples Method development of suitable imaging approaches and validation Development of smart image analysis tool
Materials a. b. c. d. 50 nm 100 nm f. 20 nm Fumed silica (a.) and silver NP (b.) spiked into tomato soup (c.) and lean chicken meat (d.), respectively. Gold NP (f.) due to stability and good contrast are used as model particles in experiments.
Methods Drying (air drying, freeze-drying, chemical drying Sedimentation of the sample by ultracentrifugation Resin embedding Freezing Blotting excess moisture off with filter paper
Influence of sample preparation on SiO 2 nanoparticles in soup
Improvement of blotting by treatment of the grid with poly-l-lysine No treatment Poly-l-lysine treated grid
Influence of sample preparation technique on particle size Argument Sample Preparation Method Air- Drying Freeze- Drying Ultracentrifugation Blotting Blotting + Poly-llysine SiO 2 in water Mean diameter 187 244 237 229 174 148 133 82 93 59 Nanoparticles % 11.8% 9.1% 15.5% 23.6% 37.3% SiO 2 in soup Mean Diameter 583 749 573 1025 432 255 593 766 211 279 Nanoparticles % 0 2% 2% 0 28%
Poly-l-Lysine, performance dependent on stabilizing agent 500nm 500nm
Recovery of nanoparticles Argument Water Buffer ph 8.0 SiO 2 water SiO 2 soup SiO 2 water SiO 2 soup Mean size (nm) 114±36 127±39 116±47 129±45 Total PNC [E8/ml] 3.22±0.55 1.62±0.43 4.05±0.59 3.47±0.53
Sample preparation method s performance Silica nanoparticles spiked in tomato soup Method Dilution factor Diluent No of particles Mean size (nm) Ultracentrifugation 1 in 500.000 Water 72 334 237 0% Ultracentrifugation 1 in 500.000 ph 8.0 4293 127 78 18% Blotting + poly-llysine 1 in 200 ph 8.0 1836 114 81 31% Blotting + gelatine 1 in 200 ph 8.0 2044 117 79 28% Blotting+alcian blue 1 in 200 ph 8.0 735 132 103 26% Nanofraction %
Liquid imaging using poly-l-lysine SiO2B in soup- consecutive images of same area a) first image, b) after 10s of exposure, c) after 30s of exposure
Liquid imaging- performance Liquid state images of a) AgNPs in meat by ClairscopeTM, b) AgNPs in meat by QuantomixTM, c) silica beads in tomato soup- ClairscopeTM, d) silica beads in water- ClairscopeTM
Resin embedding Samples encapsulated in agarose a) before embedding, b) embedded in resin capsule is tilted according to centrifugal force angle and so is AgNPs and SiO 2 NPs dispersion within it., c) TEM image of 200nm section of resin embedded AgNPs mixed wit SiO 2 NPs
On-grid ultracentrifugation of Ag NPs in water and meat 500nm
Freezing a. b. Cryo-SEM of AgNPs in meat a)frozen by insertion to the chamber, b)plunge frozen in liquid nitrogen
Conclusions Air drying and freeze drying cause spatial agglomeration and aggregation of NPs Ultracentrifugation and blotting can be used for preparation of liquid and solid food samples Use of alkaline ph stabilizes and facilitates recovery of NPs during sample processing- dilution, centrifugation Use of poly-l-lysine for liquid imaging facilitates detection and characterization of NPs Freezing in the liquid nitrogen creates unnecessary topography on the sample surface
Acknowledgments Ping Luo Ian Morrison Meg Stark Paul Beales Karen Tiede Kristian Mølhave Alistair Boxall Alan MacNicoll Qasim Chaudhry Roland Kröger Jun Yuan Edward Boyes Pratibha Gai Ian Wright Michael Walsh Gonzalo Vallejo Fernandez