an overview of new technologies in veterinary chemical ... · an overview of new technologies in...

An overview of new technologies in

veterinary chemical residue control in

food by rapid methods: from classical

to innovative technologies

1

Food labs in Crystal Balls

Future challenges in food analyses

AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015

21 ‐ 22 May 2015, Stockholm, Sweden

Valérie GAUDIN Anses – French Agency for Food, Environmental and

Occupational Health Safety

Laboratory of Fougeres

What are the biological

techniques available to quickly

screen for veterinary drugs in

food of animal origins?

AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015 2

Screening methods • First critical step for the control of

veterinary drugs in food,

• Confirmatory methods.

• Screening methods:

– Cheap

– Quick

– Sensitive (< 5 % of false negative results)

– Specific or with a wide spectrum detection

depending on the target analytes

– High throughput of samples.


Classical techniques

• Microbiological

– Plate tests, microplate or tube format

– Largely used all over the world

– Cost-effective

– Wide spectrum of detection

• Immunological

– ELISA, RIA, dipstick

– Usually more specific and sensitive


4

New trends: Biosensors


Bioreceptors Transducers

Antibodies

Molecular

imprinted

polymers (MIPs)

Enzymes

Aptamers

(Nucleic acids)

Whole-cell

biosensors

Electrochemical

Optical Mass

sensitive

History

• Biomedical applications started in the

1980’s

– e.g. glucose detection in blood

• Antibiotic residues: First article:

– Electrochemical biosensor for the detection of

(amphotericin B and nystatin) in 1979

• Biosensors for veterinary drugs in food in

continuous development


ELECTROCHEMICAL

DETECTOR

Measure electrical properties of the solution (production or

consomption of ions or electrons) , such an electric current or

potential

- Potentiometric

- Amperometric


Potentiometric

• Measure the potential difference between:

– an indicator and a reference electrode or 2 reference electrodes separated by a

perm selective membrane

• Electrodes:

– Most common potentiometric devices = pH electrodes

AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015 8 Stead SL, Wolodko-Cierniak KB, Richmond SF, Sharman M, Driver P, Tealeb P, Leonardova O, Purvisc D. 2011. Development

and validation of a potentiometric biosensor assay for tylosin with demonstrated applicability for the detection of two other

antimicrobial growth-promoter compounds in feedstuffs. Food Additives and Contaminants. Vol. 28, No. 7:848-859.

• Platform for developing ligand-binding assays: Multi-probe format assay for the simultaneous

detection of tylosin, spiramycin and

virginiamycin in animal feed

Multiplexed assays: simultaneous measurement of

up to 12 samples

Pre-coated screen-printed electrodes, single use

Easy sample prep

Rapid (less than 45 min)

Lab or portable field-test

CCβ = 200 µg/kg

Transferable, reliable and robust

Results comparable with LC-MS/MS

Vantix™ (UK):

Amperometric • Measure a resulting current: Applied

potential electrochemical oxidation or

reduction of an electroactive species

• Working electrode:

– a noble metal

– Carbon paste with an embedded enzyme

– a screen-printed layer covered by the

bioreceptor (SPCE)


Conzuelo F, Ruiz-Valdepeñas Montiel V, Campuzano S, Gamella M, Torrente-Rodríguez RM, Reviejo AJ, Pingarrón JM. 2014.

Rapid screening of multiple antibiotic residues in milk using disposable amperometric magnetosensors. Analytica Chimica

Acta. 820:32-38.

Gamella M, Campuzano S, Conzuelo F, Esteban-Torres M, de las Rivas B, Reviejo AJ, Munoz R, Pingarron JM. 2013. An

amperometric affinity penicillin-binding protein magnetosensor for the detection of [small beta]-lactam antibiotics in milk.

Analyst. 138:2013-2022.

• Multiplex screening of CEPHs, SULFs and TTCs in milk: Direct competitive assays using HRP-labeled tracers

3-target specific magnetic beads (MBs) modified with PBPs or antibodies

Easy sample prep

Analyzing spiked milk samples in only 5 min

Quick, simple, easy automation and miniaturization, sensitive ≤MRL

More suited for mass production than potentiometric

Promising alternative for on site analysis

SPY = sulfapyridine PBPs = penicillin binding proteins)

MASS SENSITIVE DETECTOR Response to the mass of solute passing through the detector per

unit time.

Take advantage of the piezoelectric effect: electric charge that

accumulates in certain solid materials (i.e.crystals, certain

ceramics) in response to applied mechanical stress

- Quartz Crystal Microbalances (QCM)

- Surface Acoustic Wave (SAW)


Quartz Crystal Microbalance


Karaseva NA, Ermolaeva TN. 2012. A piezoelectric immunosensor for chloramphenicol detection in food. Talanta. 93:44-48.

Karaseva NA, Ermolaeva TN. 2014. Piezoelectric immunosensors for the detection of individual antibiotics and the total

content of penicillin antibiotics in foodstuffs. Talanta. 120:312-317.

• Piezoelectric component: – quartz, tourmaline, lithium tantalate, etc

– Stable, chemically inert, excellent mechanical and aging characteristics

– Quartz - crystal coated with gold electrodes

• Label free technique: binding of molecules mass increasing

oscillation frequency of the crystal changed measured electrically

• Screening of CAP in food (meat, milk, egg, honey): Competitive format (Ab anti-CAP)

AT-cut resonator with gold electrodes

Sample prep: Milk and eggs buffer

Meat, honey extraction

Regeneration of the biolayer (reusable)

Full cycle = 20-30 minutes

Calibration curve linear between 0.5 and 100 ng/ml

Limit of detection 0.2 ng/ml (90% of the max signal)

Karaseva, N.A. and T.N. Ermolaeva, Piezoelectric immunosensors for the detection of individual antibiotics and the total

content of penicillin antibiotics in foodstuffs. Talanta, 2014. 120(0): p. 312-317.

OPTICAL DETECTOR

Surface Plasmon Resonance (SPR)

Chemiluminescence

Bioluminescence

Flow cytometry


Surface plasmon Resonance (SPR) Electromagnetic waves to monitor interactions

at a surface (early 1990s)

Real - time study of the interactions Ab/Ag,

without labeling, by exploiting the interfacial

refractive index changes associated with any

affinity binding interaction.


Biacore™, GE Healthcare Life

Sciences, Sweden

Spreeta™, Texas Instruments, USA

Autolab™ ESPRIT, Germany

Zhu, Y.;Qu, C.;Kuang, H.;Xu, L.;Liu, L.;Hua, Y.;Wang, L.Xu, C.; Simple, rapid and sensitive detection of

antibiotics based on the side-by-side assembly of gold nanorod probes. Biosensors and Bioelectronics. 2011,

26(11), 4387-4392.

Screening of gentamycin: Nanoparticles: performance of a SPR

immunosensor

Nanogolds =very small particles of pure gold

suspended in pure water

Competitive IA

UV–vis spectra Values of the aggregate

sizes at different GTM concentrations

Easy use, quick (less than 2 hours),

Sensitive

Very specific for GTM

No analyses in a complex matrix like food

Commercial systems:

Chemiluminescence • Chemiluminescent reactions:

√ labeled enzyme to catalyze a chemiluminescent reaction (e.g., horse radish

peroxidase (HRP) and alcaline phosphatase (ALP)

√ covalently label one of the complementary binding partners directly with a

chemiluminescent compound (luminol, isoluminol, acridinium esters)


14

Biosensor Analytes Matrices Highlights Assay time References

Evidence®

Investigator

(Randox, UK)

Antibiotic residues (e.g.

sulfonamides, quinolones,

streptomycin/DHS, tylosin,

ceftiofur, tetracyclines,

thiamphenicol), nitrofuran

metabolites and CAP),

coccidiostats

Milk,

muscle,

honey,

feed

Multiplex

(simultaneous

detection)

Simple extraction or

without extraction

40 samples

every 2 to 3

hours

maximum

(O’mahony

et al. 2011)

MCR 3 (r-

Biopharm,

Germany)

4 antibiotics (enrofloxacine,

sulfamethazine, sulfadiazine,

streptomycine)

Honey

Multiplex

(simultaneous

detection)

without purification or

extraction

fully automated

/ (Wutz et al.

2011)

13 antibiotics Milk 6 min (Kloth et al.

2009)

Antimicrobial arrays

• Benefits/ELISA:

– Sample prep: simple

dilution (except eggs)

– Rapid assay (45

samples < 2 h))

– Simultaneous

quantitative detection

of multiple analytes from

a single sample

– Reduced time/cost

– Low CCβ

• Disadvantages:

– Quinolones (false +)

– Closed system

(custom biochips)

AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015 15 Gaudin V, Hedou C, Soumet C, Verdon E. 2014. Evaluation and validation of biochip multi-array technology for the

screening of six families of antibiotics in honey according to the European guideline for the validation of screening

methods for residues of veterinary medicines. Food Additives & Contaminants: Part A. 31:1699-1711.

AM I (14

SULFA + TMP)

AM II ( (F)Qs,

strepto/DHS, TYLO,

CEFT, TTCs, TAF)

AM IV

(MACRO,

AMINO)

AM III (nitrofuran

metabolites, CAP)

• Platform Evidence Investigator

(Randox, UK) Biochips

Chemiluminescence detection

HRP-conjugate signal

reagent (luminol)

Competitive IA

Multiplex (multi-array screening)

1 spot per family or per AB

Matrices: milk, muscle, honey, feed

Kits ready to use (arrays)

Analytes:

O’Mahony, J.;Moloney, M.;McConnell, R.I.;Benchikh, E.O.;Lowry, P.;Furey, A.Danaher, M.;

Simultaneous detection of four nitrofuran metabolites in honey using a multiplexing biochip

screening assay. Biosensors and Bioelectronics. 2011, 26(10), 4076-4081.

Luminescent bacterial biosensors

• Bioluminescent whole-cell biosensor:

measures the luminescence change emitted by living

microorganisms

bioluminescence genes (lux) as reporters of transcriptional

responses


Virolainen NE, Pikkemaat MG, Elferink JWA, Karp MT. 2008. Rapid Detection of Tetracyclines and Their 4-Epimer Derivatives

from Poultry Meat with Bioluminescent Biosensor Bacteria. Journal of Agricultural and Food Chemistry. 56:11065-11070.

• Screening of TTCs in poultry

muscle: Escherichia coli strain TetLux

Luciferase operon under the control of a

tetracycline sensitive repressor

Lyophilised cells rehydrated

Sample prep: meat fluid extract (easy)

Quick (4 h), more than inhibition tests (18-

24h)

High throughput (96-well plate format)

Sensitive (LOD 5 ng/g DC, 7.5 ng/g, CTC,

25 ng/g TTC/OTC)

Induction coefficients = signal/background

Bead - based flow cytometric IA (1) • Particles or cells flow in a fluid stream

one by one through a sensing point

(light beam)

• Optical sensing: Intense light source

(laser or mercury arc lamp)

measures light scatter and fluorescent

signal

• Signals amplified and converted to

digital form

• Rapid physical and/or chemical

measurement per cell or particle

• Simultaneously measures multiple

characteristics of single cells

• Automated method


17

Side-scatter detector (1)

Multiple fluorescence emission

detectors (2–4)

NovoCyte Benchtop FC ACEA Biosciences, Inc.

CytoFLEX Flow Cytometer Beckman Coulter

S3e™ Cell Sorter Automated FC Bio-Rad

SpectraMax MiniMax Imaging Cytometer Molecular Devices LLC

BD Accuri™ C6 FC BD Biosciences

MOXI Flow™ Smart FC™ ORFLO Technologies, LLC

Cytell Cell Imaging System GE Healthcare Life Sciences

Guava easyCyte 6HT-2L Base System Merck Millipore

Exemples of suppliers

http://www.clinchem.org/content/46/8/1221/F1.expansion

http://www.selectscience.net/supplier/bd-biosciences/?compID=8613

Bead - based flow cytometric IA (2) • Sulfonamides in raw milk:

xMAP-technology (Luminex™, USA): Multiplex IA

Small carboxylated polystyrene microspheres internally dyed with a red and

an infrared fluorophore (varying ratio of the two fluorophores)

Simultaneously measure up to 100 different biomolecular interactions in a

single well (100 color-coded bead sets) 11 different sulfonamides

CCβ sulfadoxine < 50 µg/l (5 others); CCβ SDZ<200 µg/l (4 others)

Qualitative screening assay

Quick: Assay time < 2 hours

Automated

• BeadyPlex (Unisensor, Belgium): Multi-antibiotic in pork meat

Competitive IA

Simultaneously measure 64 ABs from 10 families (AMGL, BL, TTC,

MACRO, LINCO, CAP, FQ, SULFA)

Sensitive

High throughput 100 to 500 samples/day


de Keizer, W.;Bienenmann-Ploum, M.E.;Bergwerff, A.A.Haasnoot, W.; Flow cytometric immunoassay for

sulfonamides in raw milk. Analytica Chimica Acta. 2008, 620(1-2), 142-149.

Poster at VDRA 2014: MULTI-RESIDUE FLOW CYTOMETRY-BASED IMMUNOASSAY FOR THE SIMULTANEOUS

SCREENING OF ANTIBIOTICS, AFLATOXIN M1 AND MELAMINE IN FOOD COMMODITIES. C. SUAREZ-PANTALEON,

M. COLOMBO, A.-C. HUET, V. CHABOTTAUX, PH. DELAHAUT and B. GRANIER, Marloie, Belgium.

Conclusions Cheap Quick Sensitive Field/

porta

ble

Mutiplex High

throug

hput

Easy

sample

prep

Potentiometric x x x x x x x

Amperometric x x x x x x x

QCM x x x x x x x

SPR x x x x x x

Chemilumines

cence

x x x x x

Bioluminesce

nce

x x x x x x

CFIA x x x x x


Due to their variety and their high potential, biosensors are

probably the future of the rapid control of veterinary drugs in foods

an overview of new technologies in veterinary chemical ... · an overview of new technologies in...

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