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