1 Lièges 2004 -1- Groupe CREAB (Chimie de la Reconnaissance et Groupe CREAB (Chimie de la Reconnaissance et Etude Etude des Assemblages Biologiques) des Assemblages Biologiques) DRFMC/SI3M, CEA de Grenoble, 17 Rue des Martyrs 38054 Grenoble DRFMC/SI3M, CEA de Grenoble, 17 Rue des Martyrs 38054 Grenoble cedex 9, France cedex 9, France Pascal Pascal MAILLEY MAILLEY : [email protected], [email protected]’ +33.(0)4.38.78.44.38 Interfacing electrochemistry and DNA for the design of genosensors Interfacing electrochemistry and DNA Interfacing electrochemistry and DNA for the design of genosensors for the design of genosensors Lièges 2004 Electrochemical genosensors Electrochemical genosensors -2- DNA Biochips Principle DNA Biochips Principle ODN probes ODN probes Transduction Transduction t V Hz I... Signal Hybridisation t V Hz I... Signal Hybridisation Immobilisation Immobilisation Adsorption Adsorption Chemisorption Chemisorption Covalent Grafting Covalent Grafting Entrapment… Entrapment… Localisation Localisation Microfluidic Microfluidic Hybridisation Hybridisation t V Hz I... Signal Hybridisation Detection Detection Main goals Immobilisation Immobilisation Transduction Transduction hn hn’ Fluorophore streptavidin conjugate Biotynilated target hn hn’ Reference method : Fluorescence detection Fluorophore streptavidin conjugate Biotynilated target F F n A - n F F n A - n F F n A - n Fluorescence response in a grey level scale
19
Embed
Interfacing electrochemistry and DNA Interfacing … · 2011. 7. 12. · (chip substrate) N ss-DNA Biotin-pyrrole Peptide Streptavidin-pyrrole pyrrole tool-box ds- DNA 1.3- Electrodeposition
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
1
Lièges 2004
Ele
ctro
chem
ical
gen
osen
sors
Ele
ctro
chem
ical
gen
osen
sors
-1-
Groupe CREAB (Chimie de la Reconnaissance et Groupe CREAB (Chimie de la Reconnaissance et EtudeEtude des Assemblages Biologiques)des Assemblages Biologiques)DRFMC/SI3M, CEA de Grenoble, 17 Rue des Martyrs 38054 Grenoble DRFMC/SI3M, CEA de Grenoble, 17 Rue des Martyrs 38054 Grenoble cedex 9, Francecedex 9, France
Using Polypyrrole MatricesUsing Polypyrrole Matrices
Lièges 2004
Ele
ctro
chem
ical
gen
osen
sors
Ele
ctro
chem
ical
gen
osen
sors
-4-
ImmobilisationImmobilisation
DNA probe accessibility
Probe Integrity
Recognition properties
Recognition specificity/selectivity
Suitable with detection/transduction
11-- Electrochemical DNA Immobilisation using pyrrole and derivativeElectrochemical DNA Immobilisation using pyrrole and derivatives s
-n (2+δ) e -
-2nH+
+ nδA-n
,n A-
+
+06-0.8V vs. SCE
F
F
n A-
n
Mild conditions of immobilisation
Adressable
Routes of functionnalisation
Control of the polymer film coating
Physico-chemical activity of the matrixOther conducting polymers: PANIPANI, , PEDOTPEDOT, , PThPTh, , PJUGPJUG
3
Lièges 2004
Ele
ctro
chem
ical
gen
osen
sors
Ele
ctro
chem
ical
gen
osen
sors
-5-
Electrode
NH
NH
NH
+
Charge p-
Electropolymérisation
-n x (2.3 e-, -2H+)
n
1.11.1-- DNA Immobilisation through electrostatic interactions with PPyDNA Immobilisation through electrostatic interactions with PPy
Polypyrrole doppingPolypyrrole dopping J . Wang et al, Langmuir 16 (2000) 2269G.G. Wallace et al, Synth. Met. 123 (2001) 279A. kumar et al, Chem. Commun (2004) 820 [PEDOT]
DNADNA CounterionCounterion
Electrostatic interactionsElectrostatic interactions D.S. Minehan et al, J. Macromol. Sci. A (Pure Appl. Chem.) 38 (2001) 1245
EEpp EEoxox
Lièges 2004
Ele
ctro
chem
ical
gen
osen
sors
Ele
ctro
chem
ical
gen
osen
sors
-6-
1.21.2-- Immobilisation by postImmobilisation by post--functionnalisation of PPy matricesfunctionnalisation of PPy matricesBy covalent graftingBy covalent grafting H. Korri-Youssoufi et al, J. Am. Chem. Soc. 119 (1997) 7388
T.-Y Lee et al, Anal. Chem. 73 (2004) 5629 [PTh]
NH
OOH
NH
OO N
O
O
0,6 0,4
n
NH
OOH
NH
OO N
0,6 0,4
n
HCCT-AAG-AGG-GAG-TG
NH
OOH
NH
OO N
O
O
0,6 + 0,4
Electropolymerisation DNA grafting
By complexationBy complexationJ. Janata et al, J. Am. Chem. Soc. 125 (2003) 324
EEpp
By biological anchoringBy biological anchoringA. Dupont et al, Anal. Chim. Acta. 515 (2004) 271
4
Lièges 2004
Ele
ctro
chem
ical
gen
osen
sors
Ele
ctro
chem
ical
gen
osen
sors
-7-
pyrrole : intelligent glue for biological applications
ELECTROCHEMISTRY BIOLOGY OPTICS
(applications) (detection)
CHEMITRY (chip substrate)
N
ssss--DNADNA
PeptideBiotinBiotin--pyrrolepyrrole
StreptavidinStreptavidin--pyrrolepyrrole
pyrrolepyrrole tooltool--boxboxds- DNA
1.31.3-- Electrodeposition and biochips : the PPy toolElectrodeposition and biochips : the PPy tool--boxbox
Protein
Sugars
Microorganism
Lièges 2004
Ele
ctro
chem
ical
gen
osen
sors
Ele
ctro
chem
ical
gen
osen
sors
-8-
1.3.11.3.1-- DNA direct grafting on functionnal substrates: MICAMDNA direct grafting on functionnal substrates: MICAM™™
of DNA hybridization:of DNA hybridization:The conducting polymer approachThe conducting polymer approach
9
Lièges 2004
Ele
ctro
chem
ical
gen
osen
sors
Ele
ctro
chem
ical
gen
osen
sors
-17-
3.13.1-- PhysicoPhysico--chemical behaviour of PPy and other conducting polymerschemical behaviour of PPy and other conducting polymers
-0,16 0 0,16 0,32 0,48
-0,16
-0,12
-0,08
-0,04
0
0,04
0,08
0,12
0,16
0,2
0,24
I (m
A c
m-2
)
E (V/ECS)
1
2nnδδ AA--nnδδ AA-- --nnδδee----nnδδee--
, δ, δ AA--, δ, δ AA--
RedRed OxOx
nnN
H nnN
H nn
δδ++N
H nn
δδ++N
H
δδ++N
H
Semi-condutor (p) Conductive (bipolaron)
BC
BV
Eg
BC
BV
σσ S cm-1
- 106
-104
-102
-1
-10-2
-10-4
-10-6
-10-8
-10-10
-10-12
-10-14
-10-16
Métaux
Semi-conducteur
Isolant
Lièges 2004
Ele
ctro
chem
ical
gen
osen
sors
Ele
ctro
chem
ical
gen
osen
sors
-18-
3.23.2-- Chronoamperometric hybridization detection at ODNChronoamperometric hybridization detection at ODN--doped Ppy filmsdoped Ppy films
Probe : (dG) 20
J . Wang et al, Anal. Chim. Acta 402 (1997) 7
Comp (dC)20
Non-comp
(dA)20 or (dT)20
dA20dT20
dC20 dC20dC20
(dG) 20 probe
(dA) 20 probe
-
+
SignalSignalReversible transient signalChange in film capacitance or conductivityCurrent until charge compensation
Comp : volume effectNon-Comp: adsorption
+0.15 V vs. Ag/AgCl
10
Lièges 2004
Ele
ctro
chem
ical
gen
osen
sors
Ele
ctro
chem
ical
gen
osen
sors
-19-
A. kumar et al, Chem. Commun (2004) 820
3.33.3-- PEDOT conductivity as probing toolPEDOT conductivity as probing tool
Probe
Comp
Non-comp
Nanopore membraneGold thin film
ss-DNA doped PEDOT
∆∆∆∆R/RR/R00
SignalSignalDecrease of the PEDOT intrinsec conductivityHindrance of ionic transferEnhanced resistance variation with the duplex lenghtNo dependance of σ on the electrolyteDynamic range : 10-8 to 10-5 g mL-1
Potential (V)
n
Lièges 2004
Ele
ctro
chem
ical
gen
osen
sors
Ele
ctro
chem
ical
gen
osen
sors
-20-
NH
OOH
NH
OO N
0,6 0,4n
HCCT-AAG-AGG- GAG-TG
H. Korri-Youssoufi et alJ. Am. Chem. Soc. 119 1997 7388
3.43.4-- PPy electroactivity as transduction vectorPPy electroactivity as transduction vector