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8/14/2019 Sistem FIA Pentru Deter Min Area Aflatoxinei M1
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AFLATOXIN M1 DETERMINATION
IN RAW MILK USING A
FLOW INJECTION IMMUNOASSAY SYSTEM
AFLATOXIN M1 DETERMINATION
IN RAW MILK USING A
FLOW INJECTION IMMUNOASSAY SYSTEM
Mihaela BadeaUniversity of Rome Tor Vergata - Italy
Mihaela BadeaUniversity of Rome Tor Vergata - Italy
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CONTENT
1. Aflatoxin M1(AFM1) problematic
2. Methods for AFM1 determination
3. Principle of Flow Injection Immunoassay (FI-IA)
4. Optimisation of enzymatic label detection
5. Optimisation of AFM1 determination
6. Analytical parameters of FI-IA method
7. Real samples analysis
8. Conclusions
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O
O
OO
O
OMe
Aflatoxin B1
O
O
OO
O
OH
OMe
Aflatoxin M1
Aflatoxins are mycotoxins produced byAspergillus flavus, A.
parasiticus and A. nomius found in corn, peanuts, grains and spices.
Aflatoxin M1 is found in milk of animals that ingested feed
contaminated with Aflatoxin B1.
Aflatoxins are classified as Group 1 cancerogens (WHO, 1987).
Aflatoxin M1 is a potent liver carcinogen.
Aflatoxin M1 is relatively stable during pasteurization, storage
and preparation of various dairy products.
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Aflatoxin M1 maximum admissible level in milk (raw milk,
milk for the manufacture of milk-based products and heat-
treated milk as defined by Council Directive 92/46/EEC, as
last amended by Council Directive 94/71/EC) is 0.05
g/Kg.
COMMISSION REGULATION (EC) No 466/2001 of 8 March 2001
setting maximum levels for certain contaminants in foodstuffs
Amendments to this regulation are also proposed to bemade with respect to aflatoxin M1 (0.025 g/kg) in infantmilk and dietary products.
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1. Aflatoxin M1(AFM1) problematic
2. Methods for AFM1 determination
3. Principle of Flow Injection Immunoassay (FI-IA)
4. Optimisation of enzymatic label detection
5. Optimisation of AFM1 determination
6. Analytical parameters of FI-IA method
7. Real samples analysis
8. Conclusions
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Method
Sample
treatment Detection
Detectionlimit (ppt)
Obs.
TLC
1. Extraction
2. Clean-up
- Visual
- Densitometric
- Fluorimetric
5 - 10
Lowrecoveries
HPLC
1. Extraction on C18 cartridge
2. Derivatization- Fluorimetric
5 - 20 - Normalphase
- Goodrecoveries
1. Extraction on C18
cartridge
- Fluorimetric 15 - Reversephase
1. Extraction onimmunoafinitycolumn
- Fluorimetric 2
- Reverse
phase
TLC and HPLC methods for AFM1 determination in milk
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ELISA methods for AFM1 determination in milk
Competition Conjugate Detection Detectionlimit (ppt) Obs
Indirect - onmicrotiter plate
AFM1 - BSA Colorimetric
(405 nm)
50 - Extraction inmethanol
Indirect in aflow - throughdevice
AFB1 - BSA Colorimetric
(450 nm)
50 - clean-up stepintegrated
Direct on
microtiter plate
AFB1 - HRP Colorimetric
(450 nm)
2 - clean-up and
evaporation
Direct onmicrotiter plate
AFM1 - HRP Colorimetric
(450 nm)
10 - centrifugation
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1. Aflatoxin M1(AFM1) problematic
2. Methods for AFM1 determination
3. Principle of Flow Injection Immunoassay (FI-IA)
4. Optimisation of enzymatic label detection
5. Optimisation of AFM1 determination
6. Analytical parameters of FI-IA method
7. Real samples analysis
8. Conclusions
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buffer
GC electrode+100 mV vs Ag/AgClTMB
H2O2
Protein G
Ag ( )+ Ag* ( ) + Ab ( )
Ag = AFM1
Ag* = AFM1 HRP
Ab = Rat Monoclonal Anti-Aflatoxin
1) E. Burestedt, C Nistor, U. Schagerlf and J. Emnus,Anal. Chem., 2000, 72, 41714177
2) M. Badea, C. Nistor, Y. Goda, S. Fujimoto, S. Dosho, A. Danet, D. Barcel, F.Ventura, J. Emnus,Analyst, 2003, 128, 849856
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1. Off-line incubation of the sample containing
AFM1 with fixed amounts of AFM1- HRP (Ag*)
and Anti-AFM1 (Ab)
2. Injection of the mixture in the flow injection
system
3. Separation of the free Ag* from the bound
Ag*Ab in the protein G column
4. Free Ag* monitoring by amperometricdetermination of HRP activity using 3,3,5,5-
tetramethylbezidine (TMB) and hydrogen
peroxide
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1. Aflatoxin M1(AFM1) problematic
2. Methods for AFM1 determination
3. Principle of Flow Injection Immunoassay (FI-IA)
4. Optimisation of enzymatic label detection
5. Optimisation of AFM1 determination
6. Analytical parameters of FI-IA method
7. Real samples analysis
8. Conclusions
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Sub H2(reduced) + H2O2 Sub (oxidised) + 2 H2OHRP
- two oxidation peaks at + 250 and + 400 mV vs Ag/AgCl
CH3
CH3CH3
CH3
NH2
NH2
TMB
G. Volpe, D. Compagnone, R. Draisci, G. Palleschi,Analyst, 1998, 123, 1303 - 1307
- a working potential of + 100 mV vs Ag/AgCl was
selected for HRP activity determination
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Carrier selection-0.05 M phosphate citrate buffer, pH 5.0
-0.05 M phosphate buffer, pH 7.4
-0.05 M phosphate buffer, pH 6.5
-0.05 M phosphate buffer, pH 6.5 with 0.1M NaCl
-0.05 M phosphate buffer, pH 6.5 with 0.1M NaCl, 10 mM MgCl2
H2O2 solution
-0.05 M phosphate buffer, pH 7.4
-0.05 M phosphate buffer, pH 6.5
-0.05 M phosphate citrate buffer, pH 5.0 with 0.1 M NaCl
TMB solution
-0.05 M phosphate buffer, pH 6.5
-Distilled water
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Optimisation of TMB concentration
TMB concentration (M)
0 5x10-4 10-3 2x10-3 2x10-3
C = 10-2 mol l-1H2O2
HRP = 0.05 IU ml-1
Flow rate = 0.9 ml min-1
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H2O2 concentration influence on HRP activity
determination
0,0 5,0x10-3
1,0x10-2
0
50
100
150
200
250
Current(nA)
H2O
2concentration (M)
cTMB = 510-4 mol l-1
HRP = 0.05 IU ml-1
Flow rate = 0.9 ml min-1
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Influence of length of mixing and reaction coils
0 50 100 150 200 250
50
100
150
200
250
300
350
400
mixing coil (for a reaction coil = 200cm)
reaction coil (for a mixing coil = 50 cm)
curren
t(nA)
Length (cm)
cTMB = 510-4 mol l-1
HRP = 0.05 IU ml-1
Flow rate = 0.9 ml min-1
C = 10-2 mol l-1H2O2
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0,00 0,02 0,04 0,06 0,08 0,100
100
200
300
400
500
600
700
0,0000 0,0005 0,00100
5
10
Cu
rrent(nA)
HRP activity (IU mL-1)
Calibration graph for HRP activity determination
cTMB = 510-4
mol l-1
HRP = 0.05 IU ml-1
Flow rate = 0.9 ml min
-1
C = 10-2 mol l-1H2O2
Mixing coil = 50 cm
Reaction coil = 200 cm
I (nA) = 3.23 + 6730.68 EA HRP (IU ml-1) ; R = 0.9992
LOD = 0.25 mU mL-1 HRP (RSD = 2.34 %, n=10)
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Selection of the Ag* (AFM1 HRP) dilution
Compromise between
High dilution
Signal sufficient high (S/N >100)
AFM1 - HRP dilution
Current(nA)
0
5
10
15
20
25
30
1/200 1/100 1/501/66
Dilution 1/200 of Ag*
RSD = 1.65 % (n=10)
Using 0.03 % (v/v) Tween 20
in carrier and H2O2 solutions
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1. Aflatoxin M1(AFM1) problematic
2. Methods for AFM1 determination
3. Principle of Flow Injection Immunoassay (FI-IA)
4. Optimisation of enzymatic label detection5. Optimisation of AFM1 determination
6. Analytical parameters of FI-IA method
7. Real samples analysis
8. Conclusions
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0 20 40 60 80 100 120
5
6
7
8
9
10
11
12
current(nA)
incubation time (min)
Influence of incubation time of Ag* with Ab
Ag *(1/200) + Ab (1ppm)
Incubation media:
PBS 0.05 M, pH = 6.5without Tween 20
Temperature:room temperature(22 1 C)
0 00 0 50 1 00 1 50 2 00
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total flow rate (ml min-1)
0,00 0,50 1,00 1,50 2,00
Influence of flow rate on Ag*Ab retaining in theprotein G column
Ag *(1/200) + Ab (1ppm)
Incubation media:PBS 0.05 M, pH = 6.5
without Tween 20
Temperature:room temperature(22 1 C)
Incubation time = 1 h
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1. Aflatoxin M1(AFM1) problematic
2. Methods for AFM1 determination
3. Principle of Flow Injection Immunoassay (FI-IA)
4. Optimisation of enzymatic label detection5. Optimisation of AFM1 determination
6. Analytical parameters of FI-IA method
7. Real samples analysis
8. Conclusions
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Aflatoxin M1(ppt)
10 100 1000 10000
60
Calibration graph for AFM1 determination
1
b
a df d
x
c
= +
+
c = IC50
Analyte concentration (ppt) at50 % binding of Ag*
50% change in signal vs.the response in the absence of the analyte
AFM1 + Ag* (1/200) + Ab (1ppm)
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The main characteristics of the FI-IA for AFM1 determination
Assay 1 Assay 2 Assay 3
Calibration points (N) 12 13 12Replicates 3 3 3
LOD a / g l-1 10.4 0.9 11.1 0.9 10.9 1.0Dinamic range b / g l-1 20 - 500 20 500 20 -500IC50 / g l-1 160.6 16.6 165.8 22.1 181.3 17.9% CV cintra-assay 3.80 4.54 4.21
% CV cinter-assay 7.90
a the analyte concentration that gives a response equal with three times the standard deviation
of the zero doseb the concentration range that led to a relative change in signal between 20% and 80 %, when
compared with the zero dose
c calculated only within the dynamic range of the response
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1. Aflatoxin M1(AFM1) problematic
2. Methods for AFM1 determination
3. Principle of Flow Injection Immunoassay (FI-IA)
4. Optimisation of enzymatic label detection5. Optimisation of AFM1 determination
6. Analytical parameters of FI-IA method
7. Real samples analysis
8. Conclusions
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0 50 100 150 200 250
0
50
100
150
200
250
FI-IAmethod
HPLC method
Comparison with HPLC method (AOAC 2000.08)- Immunoaffinity column AFLA M1 AFLATOXIN TESTING- Reversed phase HPLC analytical column Nova-Pak C18 60 , 4 m, 3.9 x 300 mm- Loop injection system (50 L)- Fluorescence detection with 365 nm excitation and 435 nm emission- Flow rate 0.8 ml min-1
- Mobile phase: H2O CH3CN CH3OH (65:25:10)
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1. Aflatoxin M1(AFM1) problematic
2. Methods for AFM1 determination
3. Principle of Flow Injection Immunoassay (FI-IA)
4. Optimisation of enzymatic label detection5. Optimisation of AFM1 determination
6. Analytical parameters of FI-IA method
7. Real samples analysis
8. Conclusions
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Work in progress
Designing of an automated FI-IA
system for Aflatoxin M1
Validation of the proposed method
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ACKNOWLEDGEMENTS
Financial support:
European Community (Contract No QLK1-CT-2001-01617)
Financial support:
European Community (Contract No QLK1-CT-2001-01617)
Research team:
Prof. Giuseppe Palleschi
Dr. Laura Micheli
Prof. Emanuele Marconi
Dr. Maria Cristina Messia
Dr. Tiziana Candigliota
Special aknowledgements to:
Prof. Jenny Emnus
University of Rome Tor Vergata - Italy
University of Molise - Italy
University of Lund - Sweden
1st t
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International Scientific CommitteInternational Scientific Committe
G. Palleschi, Italy (Symposium Chair)
A. Amine, Morocco
S. Crouch, U. S. A.
G. Evtugyn, Russia
L. Gorton, Sweden
G. Guilbault, Ireland
J. Hart, England
M. Karayannis, Greece
A. A. Karyakin, Russia
V. Magearu, Romania
J. L. Marty, France
M. Mascini, Italy
H. Mottola, U. S. A.
K. Van Staden, South Africa
Local Organizing CommitteLocal Organizing Committe
University of Rome Tor Vergata
D. Moscone
S. Alarcon
M. Badea
J. Calvo Quintana
A. Lupu
L. Micheli
S. Piermarini
F. Ricci
F. Valentini
G. Volpe
LocationLocation
The conference will be held at Villa
Mondragone, one of the most beautiful"Ville Tuscolane". Now owned by the
University it has, through the centuries,
been a theatre of important events as well
as the the residence of popes and famous
families.
Built on the ruins of the Villa of the
Quintili, who were Roman consuls, it
enjoyed its maximum splendour during the
epoch of the Borghese family in the 18th
century.
In 1981 it was sold by the Order of the
Jesuits to the University of Tor Vergata.
Villa Mondragone:
Universit degli Studi di RomaUniversit degli Studi di Roma
Tor VergataTor Vergata
KINETICS INKINETICS INANALYTICAL CHEMISTRYANALYTICAL CHEMISTRY
8th International Symposium o
8-10 July 2004
ROME, ITALY
1st announcement
Villa Mondragone
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Scientific ProgrammeScientific Programme
Deadline Schedule:
15 January: 2 announcement 15 March: Abstract submission
Pre-registration FormPre-registration Form
Last name: .......................................
First name: ......................................
Institute: ..........................................
..........................................................
Department:.....................................
Address: ............................................
Postal Code: ......................................
City: ...................................................
Country: ............................................
Fax number: .......................................
e-mail address: ...................................
88thth KAC - Conference SecretariatKAC - Conference Secretariat
Communications should be directed to:
Francesca Dominici
Nancy Downer
University of Rome Tor Vergata
Via della Ricerca Scientifica, 00133
Rome, Italy
Tel. +390672594422
Fax +390672594328
e-mail: KAC2004@scienze.uniroma2 .it
Further details, including
registration form, scientific
programme, travel and
accomodation, will be sent with
second announcement and will soon
be available on the KAC-2004 web
site
(www.uniroma2.it/dipartim/BEAT/KAC2004).
The scientific programme will include
plenary lectures, oral presentations and
poster sessions to cover the following
topics:Analytical chemistry applications
based on the kinetics of:
Electrochemical reactions
Spectrometric systems
Antigen-Antibody interaction
Redox reactions
Enzyme systems
This form can be sent either by fax: +390672594328 or by email:
KAC2004@scienze.uniroma2.it as attached file.
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