7

Enzymeamperometricelectrodes
Enzymes

Biosensors &Bioelectronics I(2012/2013)

JoseA.Garrido,garrido@wsi.tum.de

Enzymeamperometricelectrodes
Enzyme immobilization
Better contact and response
Improve enzyme stability

Loss of enzyme activity

but

Sensitivity of the electrochemical signal

Electrode reusability
Less interferences

Biosensors &Bioelectronics I(2012/2013)

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

a) Noble metals (Au, Ag, Pt) modified

electrodes:
SAM provide binding sites for
proteins
SAM length affects electron
transfer

Biosensors &Bioelectronics I(2012/2013)

JoseA.Garrido,garrido@wsi.tum.de

10

Enzymeamperometricelectrodes
Electrodes for protein voltammetry
b) Metal oxide electrodes:
direct electrochemistry of
cytochrome c

electrode modified with amine groups (positively

charged) used to bind negatively charged proteins
(such a small Fe-S protein, ferredoxins)

c) Carbon electrodes (pyrolytic graphite, glassy carbon, and diamond):

generation of hydrophilic surface oxide groups similar to conducting metal oxide electrodes
d) Surfactant film:
biomembrane-like structures: lipids
accommodate proteins in a more hydrophobic environment
stabilize many proteins and can enhance their electron transfer
and catalytic properties

Biosensors &Bioelectronics I(2012/2013)

JoseA.Garrido,garrido@wsi.tum.de

11

Enzymeelectrodes.Voltammetricmethods
a) Protein sample in solution

b) Protein sample confined to electrode

Under simply reversible ET the peak-type
CV response is not influenced by diffusion
effects

Linear diffusion in planar surfaces

versus
Steady-state conditions in microelectrode
array

Biosensors &Bioelectronics I(2012/2013)

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

As a result of the strong overlapping between electrons in

conjugate molecules

conjugated ~ 0.2-0.5 -1

e d

non-conjugated ~ 1.1-1.5 -1

e d

Biosensors &Bioelectronics I(2012/2013)

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

0.2mM H2O2 steps

-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

d distance separating acceptor and donor

d0 Van der Waals distance
G0 free energy
reorganization energy

The electrochemical insulation of the enzyme

active site by its protein shell usually precludes
the possibility of any direct ET

cytochrome oxidase

Biosensors &Bioelectronics I(2012/2013)

JoseA.Garrido,garrido@wsi.tum.de

15

Enzymeelectrodes
Some enzymes exhibit direct electron transfer with electrode supports

Incomplete encapsulation of the active center due to:

presence of channels
influence of protein orientation
nano scale morphology of the electrode
( modified metal electrodes)

Biosensors &Bioelectronics I(2012/2013)

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).

potential gradient between enzyme and electrode

shuttling of e-

mediator cycled between its reduced and

oxidized state
no side reactions with the enzyme
fast reaction with the redox center of the
enzyme
reversible electrochemistry (fast rate constant)
at the electrode

a) diffusional mediators
b) mediator-functionalized electrodes
c) mediator-modified enzymes
d) enzyme bound to polymer matrix
e) interprotein electron transfer

Biosensors &Bioelectronics I(2012/2013)

JoseA.Garrido,garrido@wsi.tum.de

17

Electrontransfermediators
a) Diffusional mediators
diffusional penetration of the mediator into the protein
shuttling of e-

short distance for electron transfer

Diffusion controlled by
hydrophobic/hydrophilic properties of enzyme
and mediator
size and shape of mediator
electrostatic interaction

Ferrocene derivatives as electron acceptors for soluble oxidases (GOx)

Fe (C5H5)2

redox potential (EM0) between +0.1V to +0.4V (SCE)

rate constant for the reaction of the
reduced active center of GOx and an
oxidized ferrocene 105 M-1s-1

Biosensors &Bioelectronics I(2012/2013)

JoseA.Garrido,garrido@wsi.tum.de

18

Electrontransfermediators
b) Mediator-functionalized electrodes
Interesting for studying affinity interactions

Au electrode

100 mM

Electrocatalytic current developed by the

C60-modified electrode in the presence of
GOx and glucose

40 mM
20 mM
0 mM
5 mV/s

Biosensors &Bioelectronics I(2012/2013)

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

Covalent attachment of electron mediators units

at the protein periphery and inner sites yields
short ET distance
e- hopping or tunneling between periphery and
active sites

Glucose Oxidase covalently modified with

ferrocene electron-relay groups
increasing loading of mediators enhances
electrical contacting, but decreases of enzyme
activity. Optimal loading (Fc-GOx) 12-13 units

Biosensors &Bioelectronics I(2012/2013)

JoseA.Garrido,garrido@wsi.tum.de

20

Electrontransfermediators
multilayer-enzyme assemblies functionalized with ET mediators

30 mM
+ 0.35 V

0 mM

The electrode oxidizes the ferrocene group and

then the ferrocene oxidized the Gox redox site

Biosensors &Bioelectronics I(2012/2013)

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

Graphite electrode modified with polyallylamine

matrix containing GOx and Fc

Biosensors &Bioelectronics I(2012/2013)

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

Electrical characteristic improved by the

incorporation of electroconductive materials
(carbon powder or metal particles)
the presence of mediators enhances electron
hopping between the enzyme and the nearest
graphite particle.

Biosensors &Bioelectronics I(2012/2013)

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

Soluble cytochromes as ET mediators

only Cyct c

Bioelectrocatalyzed reduction of O2 by COx

mediated by cyt c and cyt c551 in a multistep ET
addition of cyct c551
plus COx

Biosensors &Bioelectronics I(2012/2013)

JoseA.Garrido,garrido@wsi.tum.de

24

AmperometricDNAsensors
General DNA sensor design

Biosensors &Bioelectronics I(2012/2013)

Amperometric DNA sensor

JoseA.Garrido,garrido@wsi.tum.de

25

DirectelectrochemistrywithDNA
Electrochemical activity of nucleic acids bases
anodic oxidation

( Thymine )

( Adenine )

( Cytosine )

( Guanine )

: reduction at mercury electrodes

cathodic reduction

: oxidation at carbon electrodes

: chemically reversible red/ox
of G at mercury electrodes

irreversible cathodic reduction of CA

observed in ssDNA even at neutral pH, when suitable
salt (0.3M) screens the negative charged phosphates of
the NAs

large potentials are needed high background current

destructive process

Biosensors &Bioelectronics I(2012/2013)

JoseA.Garrido,garrido@wsi.tum.de

26

IndirectelectrochemistrywithDNA
Use of electrochemical mediators
Complexes of Ruthenium and Osmium to mediate
the electrochemical oxidation of guanine

The electrode oxidizes the reduced metal

complexes which then come into contact with DNA

Guanine residues in DNA can reduce the metal

complex, regenerating the reduced mediator

How to detect the hybridization process ?

dsDNA is more negative than ssDNA
after hybridization the oxidized metal complex (positively
charged) is attracted more efficiently to the dsDNA

Biosensors &Bioelectronics I(2012/2013)

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

Biosensors &Bioelectronics I(2012/2013)

electrostatic

JoseA.Garrido,garrido@wsi.tum.de

28

Specificredoxindicatordetection
DNA modified with osmium tetroxide bipyridine (Os,bipy)
thymine

III: reduction of A and C

Biosensors &Bioelectronics I(2012/2013)

1:

Os-modified DNA

2:

unmodified ssDNA

JoseA.Garrido,garrido@wsi.tum.de

29

DNAmediatedchargetransfer
Electrochemical detection of single-base mismatches

This method takes advantage of the electronic structure of double-helical DNA,

using intercalated redox probe molecules, to report perturbation in base stacking
CV on dsDNA-modified Au electrodes

matched dsDNA

mismatched dsDNA

DNA sequence: SH-5-AGTACAGTCATCGCG

Current flows through the well stacked DNA and
the redox intercalator DM can be either reduce or
oxidize
Stacking mismatches reduce (or even supress) the
current flowing along the DNA.

Biosensors &Bioelectronics I(2012/2013)

JoseA.Garrido,garrido@wsi.tum.de

30

DNAmediatedchargetransfer
Electrocatalysis enhanced detection
CV on dsDNA-modified Au electrodes
fully base-paired

single mismatch

Current flows through the well stacked DNA to

reduce MB+ to LB+. LB goes on to reduce
ferricyanide in solution, regenerating MB+
catalytically.

Biosensors &Bioelectronics I(2012/2013)

DNA sequence: SH-5-AGTACAGTCATCGCG

JoseA.Garrido,garrido@wsi.tum.de