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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.
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015 3
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
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015
4
New trends: Biosensors
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015 5
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
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015 6
ELECTROCHEMICAL
DETECTOR
Measure electrical properties of the solution (production or
consomption of ions or electrons) , such an electric current or
potential
- Potentiometric
- Amperometric
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015 7
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)
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015 9
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)
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015 10
Quartz Crystal Microbalance
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015 11
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
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015 12
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.
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015 13
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)
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015
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
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015 16
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
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015
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
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
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015 18
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
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015 19
Due to their variety and their high potential, biosensors are
probably the future of the rapid control of veterinary drugs in foods