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

Passive Nanostructures

● Nanoparticle ● Polymers ● Nanocoatings ● Nanostructured metals

Active Nanostructures ● Amplifiers ● Sensors ● Targeted drugs ● Adaptive structures

Nanosystems

● Guided assembling ● 3D Networks & new hierarchical architectures ● Robotics

Molecular Nanosystems

● Molecule device by design ● Atomic design; ● Emerging functions

Nanotechnology – Development and Applications

Yesterday Today & Tomorrow

Adopted from MC Roco. AIChEJ 50:890 (2004)

2000 2010 2020

4. Gen

3. Gen 2. Gen

1. Gen

today

Converging Technologies

● Nano-bio-info from nanoscale ● Cognitive technologies; ● Large complex systems (nanoscale)

Year

Advanced Nanotechnology & Consumer Products

Today

MC Roco (2011) J Nanopart Res 13:427–445

On scale, this tennis ball is the

same size in relation to earth

as a nanoparticle is to a tennis ball.

Info from: http://nanohex.org/flash/Nano_What.html

Head of pin 1 millimetre

Red Blood Cell 2.5 micrometers

Ragweed pollen 20 micrometers

Carbon nanotube 2 nanometers

= 1,000,000 nanometers = 2,500 nanometers = 20,000 nanometers = 2 nanometers

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

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

-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/

Nanotech & medicine

Consumer Products Using

Nanotech Applications

9.3 billion

6.9 billion

Source: UN, Department of Economic and Social Affairs, Population Division(2011)

Year Po

pu

lati

on

(b

illio

ns)

-world population

-meat consumption

-available crop land

-water tables

-climate change

Food Security Agro Technology

Food Science and Technology

Food production must increase

70-100% (FAO 2009 estimation)

Nanotechnology

Nanotechnology and Agro/food products

World Population Growth

Application of Nanotechnology in Agriculture Sectors

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

Precision farming

Nanosystem

Application of Nanotechnology In Food Products

Application Nanotechnology Function Nano-textured food

ingredients

Processed nano-structures

in food Novel or improved tastes, flavours,

textures

Nano-delivery

systems for nutrients

/ supplements

Nano-encapsulated

bioactive substances –

mainly additives and

supplements

Nanocarrier systems used for taste

masking of ingredients / additives

i.e. fish oils, protection from

degradation.

Organic & Inorganic

nano-sized

additives for food

manufactured in the

nanosize range Due to larger surface area, smaller

quantity would be needed.

-Surface

functionalized nano

materials

-Nano-coatings on

food contact

surfaces

-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.

Woodrow Wilson Databank http://www.nanotechproject.org/

Food Products Examples

TiO2 (anticaking agent)

Ag (FCM)

NP Ag and NP TiO2 will be used as example for sample prep and detection later

Supply Seeds Fertilizers Feed, etc

Farm Primary Production

Processing /Packaging

Distribution Network

Retail Stores

Consumers

Nanotechnology has an impact on every aspect of the food supply chain,

from how food is cultivated, produced, processed, packaged and stored.

Food and food related products incorporating nanotechnology

include nanoscale fertilizers, pesticides, food additives,

enzymes, flavourings, and food packaging materials.

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

Food

Presence of NPs in Agriculture / Food Products

and Possible Routes of Human Exposure

Food and FCMs

Feed and Vet Drugs

Fertilizer & Pesticide

Not only direct food ingestion

Residues from agriculture and

industral production can

enter into air, water, soil, etc…

Animal Plants

Air respiratory

system

Food &

Water digestive

system

Water, soil, other contact skin and membranes

"All things are poison, and nothing is without poison;

only the dose permits something not to be

poisonous.” Paracelsus (1493-1541) [pærəˈsɛlsəs]

How much is too much?

Examples:

Salt & sugar

Nano particles ???

Father of the toxicology

The need for characterization & quantification

Fadeel & Garcia-Bennett (2010)

Adv Drug Del Rev 62:362

risk =

haza

rd x

exp

osu

re

Risk Assessment Paradigm is Applicable

Challenges

complexity

dynamics

trans-disciplinarity

uncertainty

Challenges

Food Product Application Specific

Challenges

Nanotech Development and its Application in General

Both are interconnected

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;

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)

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

Risk assessment

Ch

allen

ge

s

Environ. Sci. Technol.

2012, 46, 2242

Complexity in exposure consideration

-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

Ch

allen

ge

s

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

Ch

all

en

ge

s

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) Increase in surface area, reactivity and absorption behaviour

Surface charge influence on particle stability especially in dispersions

Surface chemistry 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

Characterisation Parameters of

ENPs used in food products

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

Adopted from Peters and Bouwmeester of RIKILT

Different sample preparation for sample contains NPs’ aggregates and

agglomerates will affect the detection outcome.

Analytical methods for NPs characterization

Modified from Tiede at al. 2008. Food Additives and Contaminants 25(7) 795–821

Microscopy related techniques STEM, TEM, SEM : size, size distribution, Morphology /shape, aggregation,

structure,

AEM, CFM: mass concentration, surface chemistry SEM/TEM

ICP-MS

Elemental analysis / quantification

(metal / metaloxide NPs)

Field-Flow Fractionation

Separation / fractionation

Chromatography related techniques HDC, FFF: size distribution

ICP-MS: elemental analysis (quantitative)

Analytical methods for NPs characterization

Modified from Tiede at al.

2008. Food Additives and

Contaminants 25(7) 795–821

Spectroscopic related techniques NMR: chemical composition

XRD: aggregation, chemical composition, elemental analysis,

structure,

Centrifugation & filtration techniques UC, CFF: size distribution

Other techniques Zeta potential: surface charge, aggregation

BET: surface area & porosity

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

Ab

bre

via

tion

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

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

A method is developed and

validated for sizing and

quantifying NP Ag in

chicken meat using SP ICP-MS

-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.

Sample prep

Anal Bioanal Chem. 2014 Jan 5. [Epub ahead of print]

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

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.

EM

Anal Bioanal Chem. 2014 Jan 5. [Epub ahead of print]

EM pictures and EDX spectra of chicken digests

Anal Bioanal Chem. 2014 Jan 5. [Epub ahead of print]

The EDX spectra show that

pure silver is partly transformed

into silver sulfide

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 E

xam

ple

s 2

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

Defatting b/f NPs suspension reducing matrix interferences on the target particles

Comparison of extraction procedures

for TiO2 NPs from sunscreen

from J. Anal. At. Spectrom., 2012, 27, 1084

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

Sample Solvent Ti [mg kg1] Ti [mg kg1] Extraction efficiency replicate 1 replicate 2 [%] (standard deviation) SPF 15 Water 36.3+ 1.8 34.7 +1.7 64.0 (0.5) SPF 30 Water 66.0 +1.8 54.3 +1.8 79 (9) SPF 50 Water 113.8 +1.7 138.3 +1.8 71 (13) SPF 15 2.5% (v/v) hexane 41.6 +1.8 33.6 +1.8 68 (8) SPF 30 2.5% (v/v) hexane 72.2 +1.8 60.5 +1.8 87 (7) SPF 50 2.5% (v/v) hexane 195.8 +1.8 195.2 +1.8 110 (5) 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 concentration and extraction efficiency

were determined by ICP-MS

-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.

NPs TiO2 loss On tube wall

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

AVA: CH’NG Ai Lee

Tze Hoong CHUA

Paul CHIEW

CHEW Siang Thai

NTU: Kee Woei NG

NUS: David LEONG

RIKILT: Ruud Peters & his colleagues

LGC: Heidi Goenaga-Infante & her colleagues

Thanks