Towards the development of an amperometric immunosensor for the determination of Ochratoxin A
Introduction
World Health Organization
Mycotoxins(from fungi)
Phycotoxins(from algae)
Toxins from edible plants
Regulations to protect public health and economical
interests of producers and traders
Ochratoxin A (OTA): 7-(L--phenylalanyl-carbonyl)-carboxyl-5-chloro-8-hydroxy-3,4-dihydro-3R-methylisocoumarin
Effects on animals:nephrotoxic, hepatotoxic, neurotoxic, immunotoxic, genotoxic, teratogenic, myelotoxic and carcinogenic (in mice and rats) properties
Effects on humans:- Group 2B (possible human carcinogen)- Main target organ: kidneys. Nephropaties
Introduction
ONH
OCOOH OH
Cl
CH3
O
OTA occurs when food with a high water content is not properly dried
OTA in foodstuffs:
Introduction
Cereals (corn, barley, rice,…) and cereal products Cocoa and cocoa products Coffee (roasted or soluble) Species Dried vine fruits (currants, raisins and sultanas) Meat and meat products Beer Grape juice and must Wine
5 ppb
5-10 ppb
10 ppb
2 ppb 2 ppb
Analytical methods used for OTA determination
Extraction using organic solvents
Purification through chromatographic columns
Detection
Introduction
HPLC with fluorescence detection
GC/MS
Capillary electrophoresis with fluorescence detection (induced by laser)
Commercial kits for OTA based on Enzyme-Linked Immunosorbent Assays (ELISA)
Direct competitive assays
R-Biopharm (LOD:5 ppb)Romer Labs (2-40 ppb LOD:2 ppb)Neogen (2-25 ppb LOD:1 ppb)
Indirect competitive assays
Biosystems (1-50 ppb LOD:1 ppb)
Introduction
Our purpose…
Introduction
Immunoassaysgood selectivitygood sensitivitywide variety of applications
Wells
Electrochemical immunosensorsmore sensitivecost-effective instrumentationpossible miniaturizationportable devicepossibility of a high throughput sample
SPEs
Our purpose…
Introduction
Development of an immunosensor for OTA determination based on an electrochemical detection
Screen-printed electrode
Enzyme product
Enzyme substrate
anti-OTA PAbOTA
OTA-BSA
IgG-enzymeconjugate
anti-OTA PAb
Synthesis of OTA-BSA
OTA-BSA conjugate
N
N
NN
O
ONH
OCOOH OH
Cl
CH3
O
R1 COOH
+
N
NR1
O
NHN
+ + CO2
R1 NH
O
R2 NHN
+R2 NH2
+
BSA
protected from lightconstant stirring
10 minat room T
added dropwise
2 h constant stirring
OTA-BSA
Immunosensor
Strategy 1
Screen-printed electrode
Enzyme product
Immunosensor
Enzyme substrate
anti-OTA PAbOTA
OTA-BSA
IgG-enzymeconjugate
anti-OTA PAb
ImmunosensorImmunosensor
[OTA-BSA] = 0.2 µg/mL
[anti-OTA] = 1:100
[IgG-HRP] = 1:6000
1 h competition (10 µL anti-OTA + 90 µL OTA) 1 h incubation (IgG-enzyme conjugate)
Checkerboard on wells
Immunosensor
Saturation dilutions:- 1:1500 IgG-HRP- 1:250 IgG-AP
Approx. 80%:-1:6000 dilution of IgG-HRP-1:1000 dilution of IgG-AP
0.5, 1, 4, 8, 12 and 16 mg/mL p-NPP in 10% DEA buffer, pH 9.8
IgG-enzyme conjugate
log(dilution of IgG-enzyme conjugate)
Ab
s(re
lati
ve)
(62
0 o
r 4
05
nm
)
-5 -4 -3 -2 -1 0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
IgG-HRP
IgG-AP
[anti-OTA]= 2g/ml
Immunosensor
Colourimetric OTA detection (ELISA wells)
IC50 (IgG/HRP) = 341 µg/L IC30 (IgG/HRP) = 58 µg/L IC10 (IgG/HRP) = 10 µg/L
IC50 (IgG/AP) = 1366 µg/L IC30 (IgG/AP) = 390 µg/L IC10 (IgG/AP) = 111 µg/L
% o
f a
bs
orb
an
ce
log([OTA] (mg/ml))
-3 -2 -1 0 1 2
0
20
40
60
80
100
IgG-HRP
IgG-AP
n = 4
ImmunosensorImmunosensor
Checkerboard on SPEs
[OTA-BSA] = 0.2 µg/mL
[anti-OTA] = 1:100
[IgG-HRP] = 1:500
[IgG-AP] = 1:750
1 h competition (10 µL anti-OTA + 90 µL OTA) IgG-enzyme conjugate incubation: 1 h in milk
Immunosensor
Colourimetric OTA detection (SPE)%
of
ab
so
rba
nc
e
log([OTA] (mg/ml))
-3 -2 -1 0 1 2
0
20
40
60
80
100
120
IgG-HRP
IgG-AP
IC50 (IgG/HRP) = 7841 µg/L IC30 (IgG/HRP) = 311 µg/L IC10 (IgG/HRP) = 12 µg/L
IC50 (IgG/AP) = 2363 µg/L IC30 (IgG/AP) = 332 µg/L IC10 (IgG/AP) = 47 µg/L
n = 4
-0.3 -0.2 -0.1 0 0.1-0.35
-0.30
-0.25
-0.20
-0.15
-0.10
-0.05
0
0.05
E (V vs. Ag/AgCl)
i (m
A)
Electrochemical detectionImmunosensor
MPMS (N-methylphenazonium methyl sulfate)
MPMS + HRP + H2O2
MPMS Chronoamperometry(single-drop analysis)
2 min substrate incubationEreading = - 0.2 V for 20 sec
0.2 mM MPMS + 20 mM H2O2
in 100 mM acetate buffer with NaCl
Immunosensor
Electrochemical detection
Total system: 5523 nA (12.6 %)No IgG-HRP: 3509 nA (4.8 %)No anti-OTA: 3500 nA (5.0 %)No H2O2: 3333 nA (7.6 %)No MPMS: 1040 nA (7.1 %)
% o
f a
bs
orb
an
ce
log([OTA] (mg/ml))
-3 -2 -1 0 1 20
20
40
60
80
100
n = 3
IgG-HRP IC50 = 1089 µg/L IC30 = 234 µg/L IC10 = 51 µg/L
MPMS in solution
ConclusionsConclusions
Strategy 1 allows the colourimetric detection of OTA, using wells and SPEs, and the development of an electrochemical immunosensor for the determination of this mycotoxin.
Both IgG-enzyme conjugates, with HRP or AP, can be used. Up to now, good results are observed when working with an electrochemical mediator (MPMS) in solution for the determination of HRP.
However, work is in progress since our aim is to achieve lower limits of detection (0.05-0.1 µg/L).
IMMUNOSENSOROTHER STATEGIES
Future work
Screen-printed electrode
Enzyme product
Enzyme substrate
anti-OTA PAbOTA
IgG-enzymeconjugate
anti-OTA PAb
SAvbiotin-OTA
Strategy 2
Screen-printed electrode
Enzyme product
anti-OTA PAb
Enzyme substrate
OTA biotin-OTA
SAv-enzyme conjugate
Future work
Strategy 3
Screen-printed electrode
Enzyme product
anti-OTA PAb
Enzyme substrate
OTA-enzymeconjugate
OTA
Future work
Strategy 4
Development of an immunosensor for OTA determination Development of an immunosensor for OTA determination based onbased on QCM detectionQCM detection
Quartz
anti-OTA PAbOTAanti-OTA PAb
Avbiotin-OTA
Development of an immunosensor for OTA determination Development of an immunosensor for OTA determination based on QCM detectionbased on QCM detection
Quartz of oscillationfrequence of 27 MHz
Development of an immunosensor for OTA determination Development of an immunosensor for OTA determination based on impedimetric detectionbased on impedimetric detection
Gold electrode
anti-OTA PAb
OTA
biotin-OTA
Streptavidin-Au
Av
Random sequence
Constantregion
Constantregion
5’ 3’
ssDNA oligonucleotides
Combinatorial LibraryTarget
OTAMagnetic
Beads
Binding
Washing
Elution
AmplificationPCR
Purification of ssDNA
dsDNA
Enriched ssDNA pool
1. SELEXRound
2-n SELEXRound
Last SELEXRound
Cloning andSequencing
SELEXProcess
MB
MB
MB
MB
MB
Removal of unboundoligonucleotides
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