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Real Time Electrochemical Displacement Immunosensor
for TCA detectionMaría Viviana Duarte
2nd year PhD Student
Project thesis course
January 2004
5 W and 1 H
Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte
What?
Real Time Electrochemical Displacement Immunosensor for TCA detection - 2,4,6 trichloroanisole (TCA) detection using antiTCA-IgG
Who? MV Duarte- 2nd year PhD student. Project thesis
When?
PhD studies
Where?
BBG group – Etseq Universitat Rovira I Virgili
Why? TCA - as an indicator of contaminants responsible for cork taint
How? Our Tools- Antibodies and Electrochemical immunosensor
Cork taint incidents
Why- An introduction
Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte
2,4,6-trichloroanisole (TCA) An indicator!
Cost money to cork and wine industry
Antibodies
How- Our Tools
Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte
Immunosensors
Selective immunodetection of TCA
Displacement methodology: reagentless and fast responding devices
How- The detection
Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte
Antibody
antiTCA- IgG
150 k DA
TCA
0,2 k DA
Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte
Graphite electrode
substrate
substrate
e -
product
productHRP labelled TCA
Amperometric signalSpecific and non- specific Adsorption
Adsorption amperograms
C:\Autolab\Data\Usuaris\Viviana\2004\11 Noviembre\08\Izq rabbit, chrono ads.ixw
0 250 500 750 1000 1250 1500 1750 2000-5-0.700x10
-5-0.600x10
-5-0.500x10
-5-0.400x10
-5-0.300x10
-5-0.200x10
-5-0.100x10
0
-50.100x10
t / si /
At/seg
TCA-HRP
1- TCA- HRP Adsorption
Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte
Materials and Methods- System 1
Specific and non- specific Adsorption
Adsorption amperograms
C:\Autolab\Data\Usuaris\Viviana\2004\11 Noviembre\08\Izq rabbit, chrono ads.ixw
0 250 500 750 1000 1250 1500 1750 2000-5-0.700x10
-5-0.600x10
-5-0.500x10
-5-0.400x10
-5-0.300x10
-5-0.200x10
-5-0.100x10
0
-50.100x10
t / s
i /
A
t/seg
TCA-HRP
Graphite electrode
substrate
substrate
e-
product
productHRP labelled TCA
1- TCA- HRP Adsorption
2- TCA (analyte) addition
Displacement
TCA Hapten-HRP labelled on Clon 007 electrode
Displacement chronoamperogram
C:\Autolab\Data\Usuaris\Viviana\2004\11 Noviembre\03\EIZQ 2 CLON007, chrono DESPLAZ.ixw
0 500 1000 1500 2000 2500 3000-5-0.300x10
-5-0.250x10
-5-0.200x10
-5-0.150x10
-5-0.100x10
-5-0.050x10
0
-50.050x10
t / s
i / A
Substrate addition
TCA (analyte) additions
Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte
Specific and non- specific Adsorption
Adsorption amperograms
C:\Autolab\Data\Usuaris\Viviana\2004\11 Noviembre\08\Izq rabbit, chrono ads.ixw
0 250 500 750 1000 1250 1500 1750 2000-5-0.700x10
-5-0.600x10
-5-0.500x10
-5-0.400x10
-5-0.300x10
-5-0.200x10
-5-0.100x10
0
-50.100x10
t / s
i / A
System 1- ResultsTCA Hapten-HRP labelled on Clon 007 electrode
Displacement chronoamperogram
C:\Autolab\Data\Usuaris\Viviana\2004\11 Noviembre\03\EIZQ 2 CLON007, chrono DESPLAZ.ixw
0 500 1000 1500 2000 2500 3000-5-0.300x10
-5-0.250x10
-5-0.200x10
-5-0.150x10
-5-0.100x10
-5-0.050x10
0
-50.050x10
t / s
i / A
TCA injections started
TCP or HaptenA
Specific and non-specific Adsorption
Adsorption Amperograms
TCA- HRP on IgG electrode
Displacement chronoamperogram
Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte
1. Substrate
2. TCA-HRP
1- TCA- HRP Adsorption 2- TCA addition
Substrate
e -
product
product
Amperometric signalSpecific and non- specific Adsorption
Adsorption amperograms
C:\Autolab\Data\Usuaris\Viviana\2004\11 Noviembre\08\Izq rabbit, chrono ads.ixw
0 250 500 750 1000 1250 1500 1750 2000-5-0.700x10
-5-0.600x10
-5-0.500x10
-5-0.400x10
-5-0.300x10
-5-0.200x10
-5-0.100x10
0
-50.100x10
t / si /
At/seg
Antimouse-HRP
1- antimouse- HRP Adsorption
Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte
Materials and Methods- System 2
substrate
substrate
TCA-BSA
1- Antimouse- HRP Adsorption
2- TCA (analyte) addition
Displacement
Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte
e -
product
product
substrate
substrate
TCA Hapten-HRP labelled on Clon 007 electrode
Displacement chronoamperogram
C:\Autolab\Data\Usuaris\Viviana\2004\11 Noviembre\03\EIZQ 2 CLON007, chrono DESPLAZ.ixw
0 500 1000 1500 2000 2500 3000-5-0.300x10
-5-0.250x10
-5-0.200x10
-5-0.150x10
-5-0.100x10
-5-0.050x10
0
-50.050x10
t / s
i / A
System 2- Results
Displacement experiments are the next step!
BSA or BSA HA 5%- CoPos90%-Peg 5% - 30 MIN 37şC clon 002
adsorption - solo antimouse hrp
C:\Autolab\Data\Usuaris\Viviana\2004\11 Noviembre\29\Eizq BSA_30min37Ccl002, chrono ads amouse 1_800.ixw
0 500 1000 1500 2000 2500 3000-6-1.000x10
-6-0.750x10
-6-0.500x10
-6-0.250x10
0
-60.250x10
-60.500x10
t / s
i / A
BSA electrode
BSA or TCA-BSA 5%- Co Pos 90%-PEG 5%; 30 min 37ºC anti TCA IgG; chornoamperometric signal of antimouse HRP
TCA-BSA electrode
Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte
50 nm
Incident light Reflected light
Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte
50 nm
channel
Immobilized
antiTCA antibodyHapten A- HRP
Immobilized
antiTCA antibodyHapten 1- HRP
+
+
kon: 5,22 E+2 M-1sec-1
koff: 7,14 E-4 sec-1
koff: 4,72 E-3 sec-1
K Association=kon/koff
K A (M-1)
7,31 E+5
1,90 E+5
Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte
kon: 8,97 E+2 M-1sec-1
•SPR Characterization: The affinity of immobilized AntiTCA IgG Clon 007 with Hapten A- HRP is 3,5 times higher than with Hapten1-HRP
•Immuno-electrochemical sensor: It seems that BSA- TCA electrodes (system 2) - AntiTCA IgG–antimouse HRP adsorbed could be use to study further TCA displacements
Conclusions
With the results obtained at the moment:
Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte
Immunoelectrochemical sensor
•Continue with electrochemichal System 2 and the Displacement experiments
•Use SPR information to improve System 1
Antibody characterization- SPRContinue with the characterization of different anti TCA-IgG (clon) and their interaction with TCA – HRP conjugations, BSA- TCA conjugations, TCA, etc
Future work
Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte
Thank you for your attention
Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte
TCA
Hapten A Hapten 1
Haptens
Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte