Nanoparticles in food and beyond. Analytical methods and ...

Nanoparticles in food and beyond. Analytical methods and ‘omics for study of their occurrence and potential health effects.

ERIK HUUSFELDT LARSEN, PH.D.LEADER OF NANO-BIOSCIENCENATIONAL FOOD INSTITUTETECHNICAL UNIVERSITY OF DENMARK

E-MAIL: [email protected]

Nanoparticles

Particles having one or more dimensions in the size range of 1 to 100 nm. One nanometer (nm) is a billionth of a meter

107x 107x

0.1

1

10

100

1000

Nanoparticles in our daily lives?Diameter (nm)

Gold atom

Albumin

Carbon atom

Asbestos

Diesel exhaust

Clay

Virus

Particles with sizes betweenabout 1 and 100 nm (at leastin one dimension)

carbon nanotube

gold nanoparticles

fullerene

Engineered nanoparticles

Nanoparticles in food: benefits vs. safety

ANTIMICROBIAL PACKAGING

Degradable foils made with nano particlesthat limit bacteria.

TEXTURE

Food spreadabilityand stability improve

with nano-sizedcrystals and lipids for

better low-fat

ENHANCED NUTRIENT DELIVERY

Nano-encapsulatingimproves bioavailability of

vitamins, antioxidants, PUFs and other‘nutraceuticals’.

NEW UNKNOWNTOXIC EFFECTS

BECAUSE OF THE SMALL SIZE

UNKNOWN RISKS FOR THE CONSUMER

HEALTH

UNKNOWN RISKS FOR THE ENVIRONMENT

Refs.: Grainger and Castner, 2008

Oberdörster et al, 2007

Q: Why are properties of nanosized particles different

from corresponding bulk material?

A: High proportion of non-coordinated surface atoms

Catalytic activity

Redox activity (ROS formation)

Types of nanomaterial mentioned in 633 records of the Nano Inventory

Ref.: RIKILT and JRC, 2014. Inventory of Nanotechnology applications in the agricultural, feed and food sector. EFSA supporting publication 2014:EN-621, 125 pp.

Ref.: RIKILT and JRC, 2014. Inventory of Nanotechnology applications in the agricultural, feed and food sector. EFSA supporting publication 2014:EN-621, 125 pp.

Nanomaterials in agriculture,

feed and food applications

Challenges when working with nanoparticlesin food and biological matrices

primary particles

Aggregation

(irreversible)

Agglomeration

(reversible)

particle-surface interaction (adsorption, repulsion)

Dissolution

…speciation…

NPcore

(Bio)molecular

corona

Energy input

9

Dispersion methods (1)Standardization of the power delivered by probe sonication

27.5

27.0

26.5

26.0

25.5

25.0

Tem

pera

ture

(°C

)

00:0001-01-1904

00:01 00:02 00:03 00:04 00:05

Time (min)

Tip amplitude (µm): 180 135 90

linear fit

Set tip

amplitude

(in % or µm)

16

14

12

10

8

6

4

2

0

Del

iver

ed p

ower

(W

)

180160140120100806040200Tip amplitude (µm)

2520151050Input power (W)

Linear fita = 0 ± 0b = 0.0846 ± 0.0006

Probe sonicator calibration by

calorimetry*

*Taurozzi et al. 2011 Nanotoxicology 5:711-29.

10Metrics for nano-particle characterisationin relation to safety assessment

Variable Importance Method of analysis

Size distribution (by mass) Essential

Shape Essential

Composition Essential

Physico-chemical structure Essential

Agglomeration state Essential

Size distribution (by number) Essential

Surface area Valuable

Surface chemistry Valuable

Surface contamination Valuable

Porosity Valuable

Surface charge in suspension Valuable, but non-essen tial

Surface charge -powder Valuable, but non-essential

Crystal structure Valuable, but non-essential

AFFF-LS-

ICP-MS

Single particle ICP-MS, or TEM

Particle detection(fractogram)

Size determination(root mean square, hydrodynamic andgeometric radius)

Asymmetric flowfield flow

fractionation

(AF4)

Inductivelycoupled plasma

mass spectrometry(ICP-MS)

Optical detection(multi angle and

dynamic light scattering, UV and

fluorescence)

Particle separationaccording to their size(small NPs elute first)

Diffusion force vs. cross flow

Elemental detectionfor identification ofparticles

Quantification

0

100000

0 50 90 130 170 210 250

m/z

90Zr140Ce

138Ba

AFFF-MALS/DLS-ICP-MS platform forcharacterisation of nanoparticles in liquid suspension

12

0.2

Inlet OutletInjection

Principle of asymmetrical flow FFF

Adapted from Wyatt Europe

10 kDa membrane

Cross flow brings

LMW material to waste

0

20

6 VD

Vwtt xr ⋅⋅

⋅⋅=&

AFFF

Cell

25 cm

0.25 mm

Fraction collection for transmission electron microscopy (TEM)

100nm

DTU Food, Technical University of Denmark

Determination of Ag NP size distribution

1 2 3 4 5

1 2 3 4 5

Diameter determined bysize-calibration curve(polystyrene standardspheres)

Loeschner et. al, J. Chrom. A (2012)

43 nm

60 nm

AF4-ICP-MS of AgNPs in enzymaticallydigested chicken meat vs. pristine AgNPs

Proteinase K 1:5 40 min@37oC (60 µg enzyme/mg tissue)

� Significant nanofraction(~80%) recovered

� Formation of additional peaksred curve (0-5 min)

� Pre-elution (~ 2 min) in comparison to pristine AgNPs:

� Sizing by tR problematic �

Q: Does AgNP peak infractogram reflectdissolution (i.e. smallersize) or non-ideal fractionation behavior?

0 10 20 30 40

0

2

4

6

8

10

extr

a pe

aks

1 &

2

void

pea

k

nanoparticlepeak

AgNPs diluted with ultrapure water enzymatically digested meat

with AgNPs

Ag

mas

s co

ncen

tratio

n (n

g/m

l)

retention time (min)t0

Loeschner, et al., Anal., Bioanal. Chem. (2013), 405, 8185-8195.

Are silver NPs toxic in an animal model?

Is silver still in particleform?

Distribution withincells?

Have the particlesagglomerated?

Imaging by TEM/EDX ofthin tissue slices (50 – 100 nm).

Distribution in organs

Determination of Ag byICPMS in tissue followingashing with HNO3/HCl

Distribution withinorgans

Silver staining (as in photography) of tissueslices followed by light microscopy

http://ratguide.com/

28 days dosage via sonde in GI tract of:

AgNPs (14 nm o.d.), or

AgAc (dissolved silver; Ag+)

16

Ref.: Oberdörster et al (2007), Nanotoxicology, 1, 2-25

Toxicity of ENPs to cells by ROS-formation

Reactive oxygen species (ROS):

.O2- Superoxide

H2O2 Hydrogen peroxide

.OH Hydroxy radical

ROOH (lipid-)peroxide

Oxidative stress =

”Too much ROS”

Dynamic light scattering (DLS) andtransmission electron microscopy (TEM)

14 nm

50 nm AgNP + PVP

Stabilised silver nanoparticles

analysed by batch-mode DLS

• Approx. 10 % of Ag as Ag+

or clusters (12,5 kDa filter)

• Long-term stable (150 d)

Organ distribution of silver –silver nanoparticles vs. silver acetate

0

10.000

20.000

30.000

40.000

50.000

co

ncen

trati

on

of

Ag

(n

g/

g w

et

weig

ht)

Ag nanoparticles Ag acetate

Organ distribution of silver –silver nanoparticles vs. silver acetate

0

10.000

20.000

30.000

40.000

50.000

co

ncen

trati

on

of

Ag

(n

g/

g w

et

weig

ht)

Ag nanoparticles Ag acetate

0

1.000

2.000

3.000

4.000

5.000 Ag nanoparticles

Ag acetate

*

**

*

*

12.6 mg/kg bw/day

9.2 mg/kg bw/day

*p < 0.05

intestinal villus macrophage

Transmission electron microscopy (TEM)

Silver nanoparticle exposed rat: ileum

lysosome containing particles


Silver acetate exposed rat: ileum

lysosome containing particles particles in the basal lamina

intestinal villus particles in the basal lamina


Silver nanoparticle exposed rat: ileum

TEM+ energy dispersive X-ray spectroscopy (EDX)

What can we learn about the chemical

composition of AgNPs inside rat’s intestinal cells?

particles

bacground

particles

background

Energi (keV) Energi (keV)

Are silver NPs more harmful to rats than dissolved silver?

Reference: N. Hadrup et al, accepteret til: ” Archives of Toxicology” september 2011

Control

AgNPs

(9 mg Ag/kg bw/day)

AgAc

(9 mg Ag/kg bw/day)

NOAEL:

AgNPs:

= 9 mg Ag/kg bw/day

AgAc:

< 9 mg Ag/kg bw/day

Bodymass increase Relative mass of thymusBodymass decrease

Plasma alkaline phosphatase Plasma urea

NH3

Folie 25

NH3 Arch Toxicol. 2012 Apr;86(4):543-51. Niels Hadrup; 15.05.2012

Urine metabolome of rats following AgNP dosage:

Female rats group separately from their controls and from males

Female vehicle

Female high NP

Male vehicle

Male high NP

Females, 9 mg/kg b.w. as AgNPs

- 0.5 0.0 0.5 1.0 1.5 PC1

PC 2

0.5

0.0

Female vehicle

Female low NP

Female mid NP

Female high NP

Female Ag-acetate

Urine metabolome of rats:

Female rats group separately from their controls, but not by dosage level nor by AgNPs vs. AgAc

Control

Ag-NP lo

wAg-N

P mid

Ag-NP h

igh

Ag-ace

tate h

igh

Alla

ntoi

n (

% c

ontr

ol)

** ** *** ***

100

Contro

lAg-

NP low

Ag-NP m

idAg-

NP high

Ag-ac

etat e

high

Uric

aci

d(%

con

trol

) * ***

100

Reference: N. Hadrup et al, Journal of applied toxicology” in press

Metabolomics by LC-Q-TOF-MS:

Excretion of uric acid and allantoin

were enhanced in female rats’ urine

Biochemical interpretation:

Uric acid and allantoin may be increased due to ROS formation

caused by exposure to AgNPs and AgAc

NH4

Folie 28

NH4 Erik bemærk at med en mere konservativ statistik foreslået af revieweren så er AgAc ikke signifikant for Uric AcidNiels Hadrup; 15.05.2012

• A large and varied box of tools, and multidiciplinarycollaboration, were necessary in nanotoxicology studies

• AgNPs or AgAc were distributed equally in the rat

• Silver, irrespective of the dosage form, exists as nanoparticles (Ag2S and/or Ag2Se) in intestinal cells

• Our research indicated that the AgNPs are (partially)dissolved and re-deposit as NPs in the cells

• Toxicological experiments with rats indicated, thatAgNPs were equally or less toxic than AgAc for theinvestigated end-points

• 1000 $-question: Is it safe to recommend future use ofAgNPs in contact with food?

Summary…..

What’s next in food nanoscience?

� Scientific advancements

� Reference materials

� Advanced instrumentation

� Standardised dispersion methods of dry nanomaterials

� Sample preparation schemes

� EU legislation and new nano-definition

Food to be labelled with ”nano”

from 13/12-2014

The essentials about food labelling with ”nano”:

1. Engineered nanomaterial is intentionally producedand has one or more dimensions below 100 nm

2. The regulation foresees adaptation of the definitionin accordance with technical/scientific progress (cf. suggested number based size distribution)

3. Ingredients as engineered nanomaterialsshall be labelled with ”nano”

EU legislation and regulation?

Number-based size

distribution of AgNPs

By:

spICPMS or

EM-methods

COMMISSION RECOMMENDATION

of 18 October 2011

New definition of nanomaterial

based on number size distribution

100%

Conclusions

• The overview demonstrates:

• Physical interferences by matrix constituentsremain a challenge, and case-to-case adaptations of sample preparation are necessary

• Even lower LODs for nanoparticle size is needed

• NanoDefine and other EU projects will assist the EU Commission in etablishing and controlling newdefinition

• Silver nanoparticles exist inside rodents‘ tissues, possibly after dissolution and redeposition assulfide and selenide salts

Technical University of Denmark, National Food Institute (DTU-Food), Nano-BioScience Group

Characterisation of seleniumnanoparticles stabilized with BSA –size determination methods

AFFF-MALS-ICP-MS

RMS - root mean square radius

freeBSA

0 10 20 30 40 50 600

1000

2000

2*R

MS

(nm

)

ICP

-MS

sig

nal (

cps)

90°

LS s

igna

l

retention time (min)

0.00

0.05

0

20

40

60

80

10 100 10000

5

10

15

20

25

volu

me

(%)

hydrodynamic diameter (nm)

Fractogram

50 nm

Transmission electron

microscopy

20nm

TEM Image

Dynamic light scattering

20

Size distribution

Resumé

• A large and varied box of tools and multidiciplinarycollaboration is necessary in nanotox studies

• AgNPs or AgAc are distributed equally in the rat

• Silver, irrespective of the dosage form, exists as nanoparticles in intestinal cells

• Our research indicates that the AgNPs are (partially)dissolved and re-deposit as NPs in the cells

• Toxicological experiments with rats indicate, thatAgNPs are equally or less toxic than AgAc for theinvestigated end-points

• 1000 $-question: Is it safe to recommend the use ofAgNPs in contact with food?

Conclusions:

• Feasible? Yes

• Partial dissolution of AgNPs

• More methods and measurementsneeded for full certification

Neat AgNP suspension

AgNPs in chicken meat

Reference materials

Advanced instrumentation

Analysis of TiO2 as Ti via Ti(NH3)x clusters using triple quad technology

Q1 Q2Cell Detector

9800 cps/ppb

10 ng/mL Ti ion sol.

Q1: m/z 46-50; Q3: m/z 148-152; Cell gas: 10% NH3 in He @ ca. 2 mL/min

Agilent technologies 8800

Pristine TiO2 NPs

in water

Mouse liver

IT admin.

Mouse liver

IV admin.

Discussion:

• New software provides easy data analysis, and results were:

• Same background equivalentdiameter (BED) in pristine and biological samples because of low background at m/z 150 (1-5 counts/dwell)

• No knowledge about NPs smaller than BED value

• Some agglomeration of TiO2 NPsin biological tissue rel. to pristineBED 53 nm

Enzymatic digestionspICP-QQQ-MS of TiO2

in mouse liver


spICP-MS analysis of fractions collectedafter AF4 separation

Thermo Fischer iCAP Q ICP-Q-MS @ 3 ms/60 kdwells

Loeschner et al.,

Anal., Bioanal. Chem. (2013) (DOI) 10.1007/s00216-013-7431-y

Optimised spICP-MS analysis ofNIST RM 8013:

A. Dwell time (3-10 ms)

B. AuNP concentration (50-260ng/L)

C. Selection of threshold

A B

C

AnimalAlkaline

(spICPMS)

Enzymatic

(spICPMS)

Aqua regia

(ICPMS)

1 3368 1794 4970

2 2875 633 3170

3 2144 1433 3922

Mean 2795 1287 4021

RSD (%) 15.6 32.7 15.9

Repeatability

RSD (%)13.0 19.6 17.2

Size and concentration of AuNPs in rat tissue determined by spICPMS after TMAH or enzymatic treatment

� Both sample preparation methods give same AuNP size distribution

� Alkaline sample preparation gives more accurate quantitative results

Number-based sizedistribution, o.d. (nm)

Quantitative results (ng Au/g)

Ref.: Loeschner, K., Brabrand, M., Sloth, J.J., and Larsen, E.H., Anal., Bioanal. Chem. (2013) (DOI) 10.1007/s00216-013-7431-y

Alkaline sample preparation in summary:

• The high content of solubilised organic matter impaired AF4 separation of Ag and Au NPs, but not their ICP-MS detection

• The solubilised organic matter hampered optical detection methods:UV, MALS or DLS, precluding direct sizing information

• The effective tissue solubilisation was however, succesful for ICP-MS detection in single particle mode

Determination of silver nanoparticle sizedistribution @ 1.0 mL/min cross flow

Calibration with size standards (polymer nanoparticles)

44nm

59nm

MALS not possible for sizing

because Ag is an absorbing

Nanoparticle and DLS is insensitive

Löschner et al., J. Chrom. A (In press)

Nano-relevantfeatures:

• Dwell time 0,2-10 ms

• Large data bufferallowing 20 mins runs @ 3 ms dwell time

• High sensitivitye.g. 300000 cpsfor 1 ng/mL Au

• Low instrumental background <1 cps@ m/z 220

Source: Thermo Fischer Scientific

The iCAPq for spICP-MS

NPs in a liquid ortissue matrix

Separation of NPs and matrix

physical

AF4- Fractionation

filtration

centrifugation

chemicalchemical

(NP-selectivechemistry)

Degradation ofthe matrix

thermalchemical

Alkaline

solubilisation

Acidic

Wet ashing

enzymatic

Dilution of thematrix

Preparation of chicken meat prior tocharacterisation of Ag-NPs

Single particle-ICPMS, the principle

Nano-relevantfeatures:

• Dwell time 0,2-10 ms

• Large data bufferallowing 20 mins runs @ 3 ms dwell time

• High sensitivitye.g. 300000 cpsfor 1 ng/mL Au

• Low instrumental background <1 cps@ m/z 220

Source: Thermo Fischer Scientific

The iCAPq for spICP-MS

Particle “events” during short dwell timesin ICP-MS

3 ms Dwells

60000 dwells

Multiple

events

Single

event

Partial

event x %

Partial

event 100-x %

Duration of

an ion plume

is 200-500 us

6 ms 9 ms 12 ms

Time scan of Ag NPs in water. 50 ng/L; 3 ms dwell; cps at m/z 107 (silver)

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

0 3000 6000 9000 120001500018000210002400027000

Sig

nal hgt(

cps)

Time (ms)

Time scan

Each signal is calibrated by an external standard curve to getmass, which is converted to size by assumption of spherical shape of nanoparticle

AF4- ICP-MS of AgNPs in enzymaticallydigested chicken meat vs. pristine AgNPs

� Significant nanofraction(~80%) recovered

� Formation of additional peaksred curve (0-5 min)

� Pre-elution (~ 2 min) in comparison to pristine AgNPs:

� Sizing by tR problematic �

Q: Does AgNP peak infractogram reflectdissolution or non-ideal fractionation behavior of AgNPs?

0 10 20 30 40

0

2

4

6

8

10

extr

a pe

aks

1 &

2

void

pea

k

nanoparticlepeak

AgNPs diluted with ultrapure water enzymatically digested meat

with AgNPs

Ag

mas

s co

ncen

tratio

n (n

g/m

l)

retention time (min)t0


Summary….spICPMS

• Pros

– Easy and fast analysis with state-of.the-art ICP-MS equipment

– Accurate size and size distribution possible

– Fit for monitoring if EU recommended nanodefinition is complied with

– No losses on physical surfaces

• Cons

– Poor mass accuracy

– Method still in it’s infancy (need for dedicated sample preparation methods)

• Therefore:

– Future coupling of sp ICP-MS with separation technique such as FFF to improveNP size separation power

– Target of future coupled method:

• Food, feed and cosmetics monitoring

• Future legislative control of NPs

Agilent 8800 ICP-QQQ

55

Q1 Q2Cell Detector

56

Aim: To produce a stable aqueous suspension of the dry nanoparticles with same characteristics across laboratories

� Energy input required (e.g. by manual shaking/vortexing/milling/ultrasound bath/ultrasound probe)

� Preferably without using stabilisers

Standardisation of dispersion methods

Ball-milling with ZrO2

beads in diluted acetic acidUltrasound probe sonicationin Milli-Q water

NPs in a liquid ortissue matrix

Separation of NPs and matrix

physical

Fractionation

filtration

centrifugation

chemicalchemical

(NP-selectivechemistry)

Degradation ofthe matrix

thermalchemical

Alkaline

solubilisation

Acidic

Wet ashing

enzymatic

Dilution of thematrix

At the forefront of nano-research: Preparationof biological samples prior to NP characterisation






Catalytic activity

Redox activity (ROS formation)

Hydrodynamicdiameter, zave (by DLS) for 9 re-plicates in eachlab. on 2 days

Verdict: Room for improvement!

Cross-laboratory results for dispersion of CuO-NH3

+ followingstandardised procedure

0

0.5

1

1.5

2

2.5

3

10 15 20 25 30 35 40 45

Retention Time (min)

Au/

Rh

- R

atio

• 10 and 60 nm AuNPs in PBS were injected in rats’ tail vein

• Alkaline dissolution of liver tissue following stabilisation of AuNPs with BSA

Aqueous Au-nanoparticlesuspension (10 and 60 nm) firststabilised with BSA and then treatedwith TMAH (pH 13)

TMAH extracts of liver homogenatecontaining 10 and 60 nm AuNPs

Recoveries were 86-123% but separation was unsuccessful

1060

Ref.: Schmidt et al. Anal. Chem. (2011)

AF4-ICP-MS of AuNPs in rat‘s liverdigested with TMAH

62

Dispersion methods (3)CuO NPs with -NH3

+or -COO-

Zave (nm) PDI ζ-potential (mV)

CuO core 1408 ± 69 0.617 14.0 ± 1.2

CuO-CH2-NH3+ 301 ± 5 0.313 27.7 ± 0.5

CuO- COO- 1224 ± 95 0.442 -6.5 ± 0,3

Before you start working…..

•Get the nanoparticles into a stable suspension, but how?

64

Dispersion methods (2)Standardization of the acoustic energy

Dispersion & size of a nanomaterial in aqueous suspension depends on the

delivered acoustic energy =acoustic power and sonication time

Other testing labs should

reach same deagglome-

ration (corresponding to 5

minutes @ 13 W using

standardised procedure)


spICP-MS analysis of fractions collectedafter AF4 separation

Thermo Fisher iCAP Q ICP-Q-MS @ 3 ms/60 kdwells

Q: Do the AgNPs dissolve during enzymaticdigestion at of chicken meat?

Techniques: spICP-MS and TEM

Slight change in particle number

size distribution after enzymatic

sample prep!


AnimalAlkaline

(spICPMS)

Enzymatic

(spICPMS)

Aqua regia

(ICPMS)

1 3368 1794 4970

2 2875 633 3170

3 2144 1433 3922

Mean 2795 1287 4021

RSD (%) 15.6 32.7 15.9

Repeatability

RSD (%)13.0 19.6 17.2

Size and concentration of AuNPs in rat tissue determined by spICPMSafter TMAH or enzymatic treatment

� Both sample preparation methods give same AuNP size distribution

� Alkaline sample preparation gives more accurate quantitative results

Number-based sizedistribution, o.d. (nm)

Quantitative results (ng Au/g)

Ref.: Loeschner, K., Brabrand, M., Sloth, J.J., and Larsen, E.H., Anal., Bioanal. Chem. (2013) (DOI) 10.1007/s00216-013-7431-y

AF4-DLS-ICP-MS fractogram of gold nanoparticles in aqueous suspension

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

10 15 20 25 30 35 40 45Retention Time (min)

Au/

Rh

- R

atio

0

10

20

30

40

50

60

70

80

90

100

Hyd

rody

nam

ic D

iam

eter

(n

m)

10 nm Au NPs

30 nm Au NPs

20 nm Au NPs

60nm Au NPs

AFFF mobile phase: 0.05% SDS

0 5 100

170000

17000

17000

17000

17000

Time (min)

Au Standard

Au NP 10 nm

Au

(cps

) Au NP 20 nm

Au NP 30 nm

Au NP 60 nm

Noise characteristics of ICP-MS detection of 0.5 ng/mL Au NPs @ 4 sizes (100 ms dwell time) and ionicstandard

Ref.: Schmidt et al. Anal. Chem. (2011)

Statement: Life is quite easywhen working with stable nanoparticles in aqueoussuspension

"Nanoparticles in Food: Analytical methods for detection andcharacterisation" http://www.nanolyse.eu

Pristine silver nanoparticles spikedto blank chicken meat as sample,

then:

Subjected to enzymatic digestionfor 40 minutes @ 37 oC

Aim: Analyse and study fate of Ag NPs by size

separation using AF4 and ICP-MS detection

Optimized AF4 fractionation of pristineAgNPs in aqueous suspension

For further details of AF4 optimisation see: Loeschner et. al, J. Chrom. A (2012)

Cross flow rate of carrier,

50 mM HN4HCO3 (mL/min)

1 mL/min for optimum selectivity and analysis time

Nanoparticles in food and beyond. Analytical methods and ...

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