Real Time Electrochemical Displacement Immunosensor for TCA detection María Viviana Duarte 2nd year...

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


2,4,6-trichloroanisole (TCA) An indicator!

Cost money to cork and wine industry

Antibodies

How- Our Tools


Immunosensors

Selective immunodetection of TCA

Displacement methodology: reagentless and fast responding devices

How- The detection


Antibody

antiTCA- IgG

150 k DA

TCA

0,2 k DA

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


Materials and Methods- System 1

Specific and non- specific Adsorption



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


Specific and non- specific Adsorption



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



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



1. Substrate

2. TCA-HRP

1- TCA- HRP Adsorption 2- TCA addition

Substrate

e -

product

product

Amperometric signalSpecific and non- specific Adsorption



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


Materials and Methods- System 2

substrate

substrate

TCA-BSA

1- Antimouse- HRP Adsorption

2- TCA (analyte) addition

Displacement


e -

product

product

substrate

substrate

TCA Hapten-HRP labelled on Clon 007 electrode



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


50 nm

Incident light Reflected light


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


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:


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


Thank you for your attention


TCA

Hapten A Hapten 1

Haptens


Real Time Electrochemical Displacement Immunosensor for TCA detection María Viviana Duarte 2nd year...

Documents

Transcript of Real Time Electrochemical Displacement Immunosensor for TCA detection María Viviana Duarte 2nd year...