An overview of new technologies in veterinary chemical residue control in food by rapid methods: from classical to innovative technologies
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An overview of new technologies in
veterinary chemical residue control in
food by rapid methods: from classical
to innovative technologies
Valérie GAUDIN
Anses – French Agency for Food, Environmental and
Occupational Health Safety
Laboratory of Fougeres
Food labs in Crystal Balls
Future challenges in food analyses
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015 1
21 ‐ 22 May 2015, Stockholm, Sweden
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.
3
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015
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
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015
New trends: Biosensors
Bioreceptors Transducers
Antibodies Enzymes Mass
Optical
sensitive
Molecular
Aptamers
imprinted
(Nucleic acids)
polymers (MIPs) Electrochemical
Whole-cell
biosensors
5
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015
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
6
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015
ELECTROCHEMICAL
DETECTOR
Measure electrical properties of the solution (production or
consomption of ions or electrons) , such an electric current or
potential
- Potentiometric
- Amperometric
7
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015
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
• Platform for developing ligand-binding assays: Vantix™ (UK):
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
Stead SL, Wolodko-Cierniak KB, Richmond SF, Sharman M, Driver P, Tealeb P, Leonardova O, Purvisc D. 2011. Development 8
AOAC
and validation of a Europe
potentiometric ‐ NMKL
biosensor assay ‐for
NordVal
tylosin withInternational, Symposium
demonstrated applicability 2015 of two other
for the detection
antimicrobial growth-promoter compounds in feedstuffs. Food Additives and Contaminants. Vol. 28, No. 7:848-859.
Amperometric
• Measure a resulting current: Applied PBPs = penicillin binding proteins) SPY = sulfapyridine
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)
• 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
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 9
AOAC
amperometric affinity Europe ‐protein
penicillin-binding NMKL ‐ NordValforInternational,
magnetosensor Symposium
the detection of [small beta]-lactam 2015
antibiotics in milk.
Analyst. 138:2013-2022.
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 10Quartz Crystal Microbalance
• 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 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.
11
Karaseva, N.A. andAOAC Europe
T.N. Ermolaeva, ‐ NMKLimmunosensors
Piezoelectric ‐ NordVal International,
for the detection ofSymposium 2015
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
12
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015Surface plasmon Resonance (SPR)
Electromagnetic waves to monitor interactions Commercial systems:
at a surface (early 1990s) Biacore™, GE Healthcare Life
Real - time study of the interactions Ab/Ag, Sciences, Sweden
without labeling, by exploiting the interfacial Spreeta™, Texas Instruments, USA
refractive index changes associated with any
Autolab™ ESPRIT, Germany
affinity binding interaction.
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
Zhu, Y.;Qu, C.;Kuang, H.;Xu, L.;Liu, L.;Hua, Y.;Wang, L.Xu, C.; Simple, rapid and sensitive detection of 13
antibiotics based on the side-by-sideAOAC
assemblyEurope ‐ NMKL
of gold nanorod ‐ NordVal
probes. International,
Biosensors and Bioelectronics. Symposium
2011, 2015
26(11), 4387-4392.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)
Biosensor Analytes Matrices Highlights Assay time References
Antibiotic residues (e.g.
sulfonamides, quinolones, Multiplex
Milk, 40 samples
Evidence® streptomycin/DHS, tylosin, (simultaneous
muscle, every 2 to 3 (O’mahony
Investigator ceftiofur, tetracyclines, detection)
honey, hours et al. 2011)
(Randox, UK) thiamphenicol), nitrofuran Simple extraction or
feed maximum
metabolites and CAP), without extraction
coccidiostats
4 antibiotics (enrofloxacine, Multiplex
(Wutz et al.
sulfamethazine, sulfadiazine, Honey (simultaneous /
MCR 3 (r- 2011)
streptomycine) detection)
Biopharm,
without purification or
Germany) (Kloth et al.
13 antibiotics Milk extraction 6 min 14
2009)
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015
fully automatedAntimicrobial arrays
• Platform Evidence Investigator
(Randox, UK)
• Benefits/ELISA:
Biochips
Chemiluminescence detection – Sample prep: simple
HRP-conjugate signal dilution (except eggs)
reagent (luminol) – Rapid assay (45
Competitive IA samples < 2 h))
Multiplex (multi-array screening) – Simultaneous
1 spot per family or per AB quantitative detection
Matrices: milk, muscle, honey, feed of multiple analytes from
Kits ready to use (arrays) a single sample
Analytes: – Reduced time/cost
AM II ( (F)Qs,
AM I (14 strepto/DHS, TYLO, – Low CCβ
SULFA + TMP) CEFT, TTCs, TAF) • Disadvantages:
AM IV
AM III (nitrofuran – Quinolones (false +)
(MACRO,
metabolites, CAP) AMINO) – Closed system
O’Mahony, J.;Moloney, M.;McConnell, R.I.;Benchikh, E.O.;Lowry, P.;Furey, A.Danaher, M.;
(custom biochips)
Simultaneous detection of four nitrofuran metabolites in honey using a multiplexing biochip
screening assay. Biosensors and Bioelectronics. 2011, 26(10), 4076-4081.
Gaudin V, Hedou C, Soumet C, Verdon E. 2014. Evaluation and validation of biochip multi-array technology for the 15
screening of six AOAC antibiotics‐inNMKL
families ofEurope ‐ NordVal
honey according to theInternational,
European guidelineSymposium
for the validation2015
of screening
methods for residues of veterinary medicines. Food Additives & Contaminants: Part A. 31:1699-1711.Luminescent bacterial biosensors
• Bioluminescent whole-cell biosensor:
measures the luminescence change emitted by living
microorganisms
bioluminescence genes (lux) as reporters of transcriptional
responses
• Screening of TTCs in poultry
muscle: Induction coefficients = signal/background
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)
Virolainen NE, Pikkemaat MG, Elferink JWA, Karp MT. 2008. Rapid Detection of Tetracyclines and Their 4-Epimer Derivatives
16
from Poultry MeatAOAC Europe ‐Biosensor
with Bioluminescent ‐ NordVal
NMKL Bacteria. International,
Journal Symposium
of Agricultural and Food Chemistry.2015
56:11065-11070.Bead - based flow cytometric IA (1)
• Particles or cells flow in a fluid stream Side-scatter detector (1)
Multiple fluorescence emission
one by one through a sensing point detectors (2–4)
(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 http://www.clinchem.org/content/46/8/1221/F1.expansion
• Simultaneously measures multiple Exemples of suppliers
NovoCyte Benchtop FC ACEA Biosciences, Inc.
characteristics of single cells CytoFLEX Flow Cytometer Beckman Coulter
• Automated method 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 17
AOAC Europe ‐ NMKL ‐ NordVal International, Symposium 2015Bead - 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 SDZConclusions
Cheap Quick Sensitive Field/ Mutiplex High Easy
porta throug sample
ble hput 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 x x x x x
cence
Bioluminesce x x x x x x
nce
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
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