TopTop--downdown trendstrends in food in food analysisanalysis: : towardstowardsnanotechnologiesnanotechnologies

Rosangela Marchelli

Department of Organic and Industrial ChemistryFaculty of AgricultureUniversity of Parma

EU EU WhiteWhite PaperPaper on Food on Food SafetySafety 20002000

•• ““……therethere hashas beenbeen anan enormousenormousdevelopmentdevelopment, in the , in the pastpast decadesdecades, , bothboth in in the the methodsmethods of food production and of food production and processing and the processing and the controlscontrols requiredrequired totoensureensure thatthat acceptableacceptable safetysafety standardsstandards are are beingbeing metmet””

Methods ofanalysis

Food safetyand quality

Regulations

www.nugo.org

Top-down and bottom-up approaches“Top-down” – building something by starting with a larger

piece and carving away material (like a sculpture)

“Bottom-up” – building something by putting together smaller pieces (like building a car engine).

AFM tip, used to manipulate, image and measure atomic scale

features.

DNA “origami”

Why is “micro and nano” good?

• Portable• Lighter

• Faster

• Lower environmental impact (reagents/solvents)• Cheaper• More energy efficient• Electronic reading allowing smart communication strategies• Different properties at very small scale

•• MethodsMethods ofofmolecularmolecular biologybiology

•• NanotechnologiesNanotechnologies•• ChromatographyChromatography

•• SpectroscopySpectroscopy

GMOsGMOsFood Food allergensallergens

Food Food AuthenticityAuthenticity

DNADNA

New methods based on genomicsNew methods based on genomics

Food Food MicrobiologyMicrobiology

NH

N

OB

O

n

O OP

O

B

O

O n

** *

*

DNA PNA

Light UpLight UpProbesProbes

SPR SPR

MicroarraysMicroarrays

HPLCHPLCCECE

PNA PNA probesprobes forfor food DNA detectionfood DNA detection

BeaconsBeacons

ColorimetricColorimetricteststests

High High techtech LowLow techtech

CDCD

The microarray The microarray technologytechnology approachapproach

MULTIPLEX PCR ANALYSIS

PNA PROBE LIBRARYDEDICATED TO FOOD

Deposition

Hybridization

MICROARRAY

3. Hybridization with the target DNA3. Hybridization with the target DNA

MICROARRAYSMICROARRAYS1. Probe design and synthesis1. Probe design and synthesis2. Probe deposition2. Probe deposition

PNA array

GMOs: development of a PCR multiplex

Germini A., Zanetti A., Salati C., Rossi S., Forre C., Schmid S., Fogher C., Marchelli, R. Journal of Agricultural and Food Chemistry, 2004, 52, 3275-3280

Specificity Specificity hybridisationhybridisation testtest

Germini A., Rossi S., Zanetti A., Corradini R., Fogher C., Marchelli R. Journal of Agricultural and Food Chemistry, 2005, 53, 3958-3962.

1.8 mm

0.8 cm

In every sub-array: more than 1 thousand spots

MZ SL RR CPMZ SL RR CP

1) Soy seedMZ SL RR CP

2) Lecithin

1 2

RR 125 bp Lectin157 bp

PNA chip PNA chip forfor RRRR--soysoy productsproducts

Detection of allergens in commercial products:hazelnut (Cor a1) and peanut (Ara h2)

PCR analyses performed on several foodstuff claiming to contain or to not contain hazelnut as ingredient or possible contaminants.

Chocolate cream with hazelnut11

Chocolate for topping10

Milk chocolate9

Biscuit topped with cocoa cream and milk chocolate8

Hazelnut wafer7

Cocoa and vanilla wafer6

Snack with cereals and cocoa5

Müesli snack with cocoa4

Müesli snack with milk chocolate3

Breakfast cereals with chocolate and hazelnut2

Breakfast cereals1

ProductSample

Chocolate cream with hazelnut11

Chocolate for topping10

Milk chocolate9

Biscuit topped with cocoa cream and milk chocolate8

Hazelnut wafer7

Cocoa and vanilla wafer6

Snack with cereals and cocoa5

Müesli snack with cocoa4

Müesli snack with milk chocolate3

Breakfast cereals with chocolate and hazelnut2

Breakfast cereals1

ProductSample

Hazelnut Cor a 1 (156bp) and PeanutAra h 2 (201bp) amplicons.

Limit of detection : 50pg DNA.

50 B 1 2 3 4 5 6 7 8 9 11bp50 B 1 2 3 4 5 6 7 8 9 1150 B 1 2 3 4 5 6 7 8 9 11bp

PNA PNA -- microarraymicroarray detection of hazelnut and detection of hazelnut and peanut DNA in commercial productspeanut DNA in commercial products

CPAPH

Sample 1. Breakfast cereals Sample 1. Breakfast cereals Sample 3. Muesli snackSample 3. Muesli snack

Rossi S., Scaravelli E., Germini A., Corradini R., Fogher C., Marchelli R. European Food Research Technology, 2006, 223, 1-6

CPAPH

50 B 1 2 3 4 5 6 7 8 9 11bp50 B 1 2 3 4 5 6 7 8 9 1150 B 1 2 3 4 5 6 7 8 9 11bp

PNA Cor PNA Ara PNA Pru Control

PNA PNA probesprobesspecificityspecificity

Detection Detection limitlimit50fmol50fmol

• Cor a 1 Hazelnut

• Ara h 2 Peanut

• Pru du 2.1Almond

Detection of hazelnut oil (5%) in extra-virgin olive oil

R

GCGAAArbequina

RCGAANocellara etnea

GCRRABosana

GCRAMoraiolo

GCGATOgliarola leccese

RCGAANocellara belice

RCGAALeccino

GCGAAFrantoio

GCGAACoratina

GCGAACarolea

GSRRACanino

GCGAABiancolilla

34519818312060

R = T o CR = T o C

S = G o CS = G o C

AuthenticityAuthenticity: Olive oil : Olive oil cultivarscultivars

SNPs in the Actin gene

CultivarCultivar

“Ogliarola leccese” “Canino”

Olive variety identification by PNA microarrays

“Frantoio”

DNADNA T A G C CP

PNAPNA A 60 T 60 C198 G198 CP

DNADNA T A G C CP

PNAPNA A 60 T 60 C198 G198 CP

DNADNA T A G C CP

PNAPNA A 60 T 60 C198 G198 CP

NOROVIRUS

Caliciviridae family: single strand RNA virus

23 milion cases per year estimated in USA

40% food-transmitted

water, mytilidae, fruits, vegetables

Main responsible for non bacterial gastroenteritis

Not cultivable in vitro

Microarrays for Norovirus

Deposition control

GIGII

NoV GII_sample 1

Deposition control

GIGII

NoV GII_sample 2

GI_compl_Cy5 (10 nM)

Depositioncontrol

GIGII

GI_compl_Cy5 (10 nM)

Depositioncontrol

GIGII

GII_compl (10 nM)

Depositioncontrol

GIGII

GII_compl (10 nM)

Depositioncontrol

GIGII

d) e)

GI_compl_Cy5 (90 nM) + GI_compl_Cy5 (10 nM)

Depositioncontrol

GIGII

GI_compl_Cy5 (90 nM) + GI_compl_Cy5 (10 nM)

Depositioncontrol

GIGII

h)

PCR products

Oligonucleotides

In collaboration with the Virology Unit of the University of Parma

Quencher

- +

Fluorophor

carboxyfluoresceinDabcyl

DNA

- +

PNA BEACON PNA BEACON (STEMLESS)OOH OH

OO

O NH

O

O

NH

O

PNA NH O

NH3+

NH

O

NH

NH2

O

NN

N

PNA BEACON FOR ROUNDUP READY-SOY

400 450 500 550 600 650 700

λ (nm)

100

200

300

400

500

600

700

F (A

.U.)

λEC=497 nm λEM=520 nm

PNA-DNA (FULL MATCH)

PNA BEACON

PNA-DNA (NOT COMPLEMENTARY)

Negative control

Beacon -DNA

(F/F0)max = 6.5

Unlabelled DNAArray with PNA beacon

PNA array

LabelledDNA

# Towards a complete “lab on a chip” system

Amplification Area using PCR

Separation Area

Detection Area

Sample inlet

The “Lab on a chip” system

Surface Plasmon Resonance

Detectorphotodiode

Typical reflectivity scan (R versus incident angle θ) before(full curve) and after the deposition of a thin film (brokencurves).

Scheme of PCR-free detection of genomic DNA using a RNAseHamplification of the SPR signal depletion. Detection limit of 10-15M was attained

T.T. Goodrich, H.J. Lee, R.M. Corn J. AM. CHEM. SOC. 2004, 126, 4086-4087

PCR-free DNA Detection

1fM oligo

Genomic DNA

Au

Lomant

Au Au

Au

PNA

Buffer

SPR microfluidic platform based onPeptide nucleic acids (PNAs)

Au

Lomant

Au Au

Au

PNA

Buffer

0 500 1000 1500

0

2

4

%R

Time (sec.)

PNA1μMPNA 0.5μM

PNA 0.1μMCoverage: 5x1012 molecule cm-2

250 μm wide80 μm deep

In collaboration with G. Spoto(University of Catania)

SPR microfluidic platform based onPeptide nucleic acids (PNAs)

5’-LL-AAACCCTTAATCCCA-3’ PNA-15mer

3’-TTTGGGAATTAGGGTTTTTTTTTTCGTCGAATAGCA-5’ssDNA-36mer-match

NPs

NPs5’-GCAGCTTATCGT-3’-BiotinssDNA-12merC6Biotin

SPR microfluidic platform based onPeptide nucleic acids (PNAs)

PEG

0 500 1000 1500 2000 2500 3000

0

3

6

9

%R

Time (sec.)

10pM

Control

match

PEG

SPR microfluidic platform based onPeptide nucleic acids (PNAs)

Prion protein detection in attomole amounts

Onisko et al., J.Am.Soc.Mass Spec., 2007, 18, 1070-1079

Prion protein

Reduction, alkylation, digestionnanoLC/MS/MS

Detection of the specific peptideVVEQMCTTQYQK

Zeptomole and attomole detection of biotin-peptides by a dot-blot gold nanoparticle

immunoassayHou S.Y. Et al., Analytical Chemistry, 2007, 79, 980-985

peptide sequence: SGQSWRPQGRFG

Detection of biotin-peptide with silverenhancement.

(A) Dot images for different levels of the biotin-peptide after silver enhancement.

B) Detection curve for the biotin-peptide

100 zeptomoles

Conclusions

• The trend in food analysis is to devise micro(nano) systems, which can provide affordable, portable, fast and user friendly equipments

• Nanotechnologies allow to push down the detection limits almost to “nothing”

• How low should the legal limits be set in food analysis ?

Acknowledgments

Roberto CorradiniStefano SforzaGianni GalavernaArnaldo DossenaTullia TedeschiAndrea GerminiChiara Dall’Asta

Stefano RossiElena ScaravelliAndrea FacciniFilbert TotsinganAlessandro AccettaAlessandro CalabrettaAlessandro TonelliValeria CavatortaMattia Mangia

RR15

RR13

RR11

SPFM image of the array after hybridisation with 1% RR125 GMO target solution

SL15

SL13

SL11

MisRR15

RR15

RR13

RR11

MisRR13

MisRR11

SL15

SL13

SL11

MisRR15

RR15

RR13

RR11

MisRR13

MisRR11

SPM image of Array B against air. All Parma PNAs (1μM probe concentration)

Surface Plasmon Microscopy

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0 5 10 15 20t / min

A 2

60 /m

V

-30

20

70

120

170

220

0 5 10 15 20t/min

F /m

V

(i)

(ii)(iii)

(iv)

(v)

b)

a)

c)

d)

b)

a)

c)

d)

(iv)(i)

Figura 14. Tracciato IE-HPLC con detector a fluorescenza (lex = 497 nm, l em = 520 nm, sinistra) e UV (260 nm, destra) relativo a : a) amplificato di PCR da solo; b) beacon RR-soia (1 mM) solo; c) beacon RR-soia (1 mM) + amplificato di PCR aspecifico; d) beacon RR-soia (1 mM) + amplificato di PCR specifico. I vari picchi sono statiidentificati come: i) beacon e componenti della reazione di PCR; ii) prodotti di amplificazione aspecifici (primer-dimers); iii) prodotto di PCR 79-mer (soia RR) iv) ibrido PNA beacon-DNA; v) prodotto di PCR aspecifico da 201bp (controllo negativo). ). Colonna: TSK-gel DNA NPR; eluenti: A = TRIS 0.02M, pH = 9.0, B = NaCl 1M in eluent A. Gradiente lineare: from 100%A to 100% B in 20 min; flusso: 0.5 mL/min.

Aflatoxins: FIAlab PMT-FL Detector (Fluorescence In-flow Assay)

0

10000

20000

30000

40000

50000

60000

70000

0 20 40 60 80 100 120

AFM1 (ppt)I (

u. a

.)

Safe < 25 ppt

Alert 25 – 50 ppt

Risk > 50 ppt

cuvette

band-pass filter

band-pass filter

PMT detector

LEDsource cuvette

band-pass filter

band-pass filter

PMT detector

LEDsource

LED: 360 nmBand-Pass Filter maximum: 450 nm

A portable fluorometer for the rapid screening of M1 aflatoxinC. Cucci, A.G. Mignani, C. Dall’Asta, R. Pela, A. DossenaSensors and Actuators B, 2007, in press

Future implementation: fiber optics.

LightUp® ProbesPNA Thiazole Orange (TO)

0

18

36

54

72

90

108

126

144

162

500 550 600 650 700Wavelenght (nm)

Fluo

resc

ence

oligo•NoGII_12mer_TO

79mer•NoGII_12mer_TO

NoGII_12mer_TO

Fluorescence emission spectra of free NoGII_12mer_TO (green line) and PNA_12mer_TO in the presence of non-complementary (blue line) and complementary (red line) DNA oligonucleotide. Samples were analyzed at a concentration of 1 µM. Excitation wavelength: 470 nm. All samples were heated at 95 °C for 3 min and spectra were acquired after 5 min at 45 °C

DUPLEX STABILITY:DUPLEX STABILITY:PNA-DNA >>DNA-DNA

POINTPOINT--MUTATION RECOGNITION:MUTATION RECOGNITION:PNA-DNA > DNA-DNA

10-mer: Tm = 50°C Tm = 35°C

15-mer: Tm drop with a mismatch Tm: 69°C -> 56°C Tm: 53°C -> 49°C

IdentificationIdentification of of specificspecific DNA DNA sequencessequences bybyPeptidePeptide NucleicNucleic AcidsAcids ((PNAsPNAs))

NH

N

OB

O

n

O OP

O

B

O

O n

** *

*

DNA PNA

0

50

100

150

200

250

0 2 4 6 8 10 12 14t (min)

F (A

.U.)

y = 42483x + 30291R2 = 0.9997

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

0 20 40 60 80 100 120

Conditions: column TSK-gel DEAE-NPR 4.6mm x 7.5cm. Gradient elution from 100%A (tris, pH= 9.0) to 100% B (1M NaCl in eluent A) in 20 min. Fluorescence detector (λex =497 nm λem=520 nm).Flow rate: 0.5 mL/min

Quantitative analysis of unlabelled DNA by RR-beacon/IE HPLC

100 nM

60 nM

10 nM1 nM

GMOs

Minunni M., Tombelli S., Fonti J, Spiriti M.M., Mascini M., Bogani P., Buiatti M.Journal of the American Chemical Society, 2005, 127, 7966-7967

PCR-FREE DNA analysis with DNA probes

35S genomic DNA detection from GM Nicotiana glauca, by piezoelectric sensing

- 1 0

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

0 1 0 2 0 3 0

t ( m i n )

F.I.

(mV)

Full match (Ogliarola, 13mer)

Mismatch (Canino e altri, 13mer)

PNA beacon

FluoFluo--GluGlu--xx xxxxxx AA xxxxxx xxxxxx--LysLys--LysLys((DabcylDabcyl))--NHNH22A 60A 60 (11mer)(11mer)

Measures:

AffinityKineticsConcentrationArraying/Imaging ispossible

Bottom-up: building sensorson self-assembled monolayers (SAM)

Surface-PlasmonFluorescence Spectroscopy(SPFS)

Flowcell

Selective detection of RR-soy PCR