Enzymeamperometricelectrodes
Enzymes
JoseA.Garrido,garrido@wsi.tum.de
Enzymeamperometricelectrodes
Enzyme immobilization
Better contact and response
Improve enzyme stability
Electrode reusability
Less interferences
JoseA.Garrido,garrido@wsi.tum.de
Enzymeamperometricelectrodes
1. Direct electrochemical methods (unmediated electrodes)
direct exchange of electrons between the electrode and the enzyme without any electroactive
mediators.
enzymes/proteins in the vicinity of the electrode
Electrodes for protein voltammetry
JoseA.Garrido,garrido@wsi.tum.de
10
Enzymeamperometricelectrodes
Electrodes for protein voltammetry
b) Metal oxide electrodes:
direct electrochemistry of
cytochrome c
JoseA.Garrido,garrido@wsi.tum.de
11
Enzymeelectrodes.Voltammetricmethods
a) Protein sample in solution
JoseA.Garrido,garrido@wsi.tum.de
12
Enzymeelectrodes.Effectoflinkingmolecules
non conjugated linkers
conjugated linkers
e- tunnelling
d
k ET k0 e d
e- tunnelling
d
conjugated ~ 0.2-0.5 -1
e d
non-conjugated ~ 1.1-1.5 -1
e d
d 1nm
d 1nm
10-1 - 10-2
10-5 - 10-7
JoseA.Garrido,garrido@wsi.tum.de
13
Enzymeelectrodes.Amperometry
At a fixed potential, the current is measured as a function of the analyte concentration
Nanocrystalline diamond electrode modified with horseradish peroxidase
0.0
0.1mM H2O2 steps
Calibration curve
-0.6
-0.8
-1.0
-1.2
-1.4
-1.6
-1.8
Current (A/cm2)
-0.4
Current density (A/cm2)
-2
Current density (A cm )
-0.2
0.05 V vs Ag/AgCl
500
1000
1500
time (s)
Biosensors &Bioelectronics I(2012/2013)
JoseA.Garrido,garrido@wsi.tum.de
14
Enzymeelectrodes
2. Indirect electrochemical methods (mediated enzyme electrodes)
the redox-active component of most redox enzymes is encapsulated deep inside the enzyme structure
effective kinetic barrier for direct electron transfer
electron transfer
rate
k ET e d d 0 e
G 0
4 kT
cytochrome oxidase
JoseA.Garrido,garrido@wsi.tum.de
15
Enzymeelectrodes
Some enzymes exhibit direct electron transfer with electrode supports
JoseA.Garrido,garrido@wsi.tum.de
16
ElectronTransferMediators
Electrical contacting of redox enzymes can be established by using synthetic or biologically
active charge-carriers as intermediates (which are called electron-transfer mediators).
a) diffusional mediators
b) mediator-functionalized electrodes
c) mediator-modified enzymes
d) enzyme bound to polymer matrix
e) interprotein electron transfer
JoseA.Garrido,garrido@wsi.tum.de
17
Electrontransfermediators
a) Diffusional mediators
diffusional penetration of the mediator into the protein
shuttling of e-
Diffusion controlled by
hydrophobic/hydrophilic properties of enzyme
and mediator
size and shape of mediator
electrostatic interaction
Fe (C5H5)2
JoseA.Garrido,garrido@wsi.tum.de
18
Electrontransfermediators
b) Mediator-functionalized electrodes
Interesting for studying affinity interactions
Au electrode
100 mM
40 mM
20 mM
0 mM
5 mV/s
JoseA.Garrido,garrido@wsi.tum.de
19
Electrontransfermediators
c) Mediator-modified enzymes
Electrical wiring of enzymes
Chemical modification of redox enzymes with
electron mediators to enable nondiffusional ET
JoseA.Garrido,garrido@wsi.tum.de
20
Electrontransfermediators
multilayer-enzyme assemblies functionalized with ET mediators
30 mM
+ 0.35 V
0 mM
JoseA.Garrido,garrido@wsi.tum.de
21
Electrontransfermediators
d) Enzyme bound to a polymer matrix
Organic polymers provide stable biocatalyst interfaces to enzymes
Electrical contacting of immobilized enzymes:
- conducting polymer
- incorporation of mediators in the polymer film
glucose concentration
(mM)
3 mM
1 mM
0 mM
JoseA.Garrido,garrido@wsi.tum.de
22
Electrontransfermediators
enzymes in mediator-functionalized sol-gel matrices
chemically inert
tunable porosity
can be easily doped
with
enzymes,
mediators,
ferrocene mediators
JoseA.Garrido,garrido@wsi.tum.de
23
Electrontransfermediators
e) Interprotein electron transfer
Small molecular electron mediators offer little selectivity in their electronic
reactions, as they exchange e- with any acceptor or donor close to them
Proteins can be used as EM
showing high selectivity
only Cyct c
JoseA.Garrido,garrido@wsi.tum.de
24
AmperometricDNAsensors
General DNA sensor design
JoseA.Garrido,garrido@wsi.tum.de
25
DirectelectrochemistrywithDNA
Electrochemical activity of nucleic acids bases
anodic oxidation
( Thymine )
( Adenine )
( Cytosine )
( Guanine )
cathodic reduction
JoseA.Garrido,garrido@wsi.tum.de
26
IndirectelectrochemistrywithDNA
Use of electrochemical mediators
Complexes of Ruthenium and Osmium to mediate
the electrochemical oxidation of guanine
JoseA.Garrido,garrido@wsi.tum.de
27
Specificredoxindicatordetection
Interaction between small redox molecules and NAs
intercalators
( daunomycin )
Fe(CN6)4-
Ir_complex
DM
( methylene blue )
MB
Ru*
Groove-binder
electrostatic
JoseA.Garrido,garrido@wsi.tum.de
28
Specificredoxindicatordetection
DNA modified with osmium tetroxide bipyridine (Os,bipy)
thymine
1:
Os-modified DNA
2:
unmodified ssDNA
JoseA.Garrido,garrido@wsi.tum.de
29
DNAmediatedchargetransfer
Electrochemical detection of single-base mismatches
matched dsDNA
mismatched dsDNA
JoseA.Garrido,garrido@wsi.tum.de
30
DNAmediatedchargetransfer
Electrocatalysis enhanced detection
CV on dsDNA-modified Au electrodes
fully base-paired
single mismatch
JoseA.Garrido,garrido@wsi.tum.de