Transcript Electrochemistry of various cellobiose dehydrogenases

PhD Course
TOPICS IN (NANO)
BIOTECHNOLOGY
Enzyme sensors
30th June
Communication between
redoxenzyme and electrode
active site
e-
electrode
wanted electron
transfer reaction
Wanted e lectron transfer path for fundamental studies and
practical applications e.g., in biosensors (here examplified
with an oxidation reaction)
oxidis e d e n z ym e
s u bs trate
e le ctrode
2 ee le ctron tran s fe r
e le ctron tran s fe r
produ ct
redu ce d e n zym e
fundamental bio-electrochemical interest as direct electron
transfer reactions between oxidoreductases and electrodes are
seldom reported
active site
e-
=
electrode
direct electron transfer
blocked due to steric or
kinetic restrictions
Electron transfer in biosensors
 First generation
 Second generation
 Third generation
facilitate electron transfer between the redox enzyme and
the electrode with a mediator
enzyme
active site
emediator
eelectrode
electron transfer occurs in
two steps:
1: betweenenzyme and
mediator
2: betweenmediator and
electrode
Mediated electron transfer path between a redox enzyme
and an electrode
oxidised enzyme
substrate
electron transfer
product
reduced mediator
electron transfer
reduced enzyme
electrode
electron transfer
2 e-
oxidised mediator
Major groups of redox enzymes used in biosensor work
oxidase
dehydrogenase
S
O2
MEDox
S
MEDox
P
MEDred
H 2O 2
P
MEDred
peroxidase
NAD-dehydrogenase
AH
H2O2
S
NAD+
MEDred
P
NADH
MEDox
A•
AH
H2O
A•
glucose/gluconolactone
-635 mV
enzyme bound flavins/PQQ
haeme (Fe 2+/3+)
-700 -600 -500 -400 -300 -200 -100
+
NAD /NADH
-560 mV
0
peroxidases (haeme)
compound I/II
+100 +200 +300 +400 +500 +600
Optimal potential range
E/mV vs. SCE
at pH 7.0
-300
-200
-100
0
+100
optimal potential range - -200 and 0 mV
E / mV vs. Ag/AgCl
vs. Ag/AgCl (at pH 7. 0)
* low background current, low noise
* no O 2 reduction
* no (or ve ry small) oxidation of ascorbic acid
uric acid
paracetamol
etc.
Electron transfer in biosensors
 First generation
 Second generation
 Third generation
 First
generation biosensors
s u bs trate
O2
oxidase
produ ct
H2 O2
e le ctrode
2 H+ + O2
2 e-
at conventional electrodes electrochemical oxidation of H2O2
occurs at ≥ + 600 mV vs. Ag|AgCl
the system is open for interfering reactions
the response is unstable with time
Ways to reduce the potential for electrochemical
conversion of H2O2
i noble metal deposition on carbon electrodes
i Prussian Blue deposition on conventional electrodes
i peroxidase modified electrodes
i other catalysts e.g. iron phthalocyanine
noble metal (Pt, Pd, Ru, Rh) deposition on carbon electrodes
lack of selectivity - future????
oxidati on at - +200-300 mV vs . Ag|AgC l
s u bs trate
s u bs trate
O2
O2
oxidase
oxidase
produ ct
redu cti on at - 0 - -150 mV vs . Ag|AgC l
produ ct
H2 O2
2 H+
H2 O2
e le ctrode
2 e2 H+ + O2
2 ee le ctrode
2 H2 O
A carbon electrode sputtered with palladium and gold for the amperometric detection of hydrogen peroxide. Gorton, L. Anal. Chim. Acta
(1985), 178(2), 247-53
Catalytic Materials, Membranes, and Fabrication Technologies Suitable for the Construction of Amperometric Biosensors. Newman, J. D.;
White, S. F.; Tothill, I. E.; Turner, A. P. F. Anal. Chem. (1995), 67(24), 4594-9.
Remarkably selective metalized-carbon amperometric biosensors. Wang, J; Lu, F; Angnes, L; Liu,
J; Sakslund, H; Chen, Q; Pedrero, M; Chen, L; Hammerich, O. Anal. Chim. Acta (1995), 305(1-3), 3-7
Electrochemical metalization of carbon electrodes. O'Connell, P. J.; O'Sullivan, C. K.; Guilbault,
G. G. Anal. Chim. Acta (1998), 373(2-3), 261-270.
deposition of Prussian Blue and related catalysts on conventional
electrodes
+ selective electroreduction of H2O2 at around 0 mV vs. Ag|AgCl
- lack of long term stability at pH > 7.5
redu cti on at - +150 - -150 mV vs . Ag|AgC l
s u bs trate
O2
oxidase
produ ct
2 H+
H2 O2
2 ee le ctrode
2 H2 O
Prussian Blue and its analogues: electrochemistry and analytical applications. Karyakin, A. A..
Electroanalysis (2001), 13(10), 813-819
Metal-hexacyanoferrate films: A tool in analytical chemistry.
de Mattos, Ivanildo Luiz; Gorton, Lo. Quimica Nova (2001), 24(2), 200-205
peroxidase modified electrodes
of great bioelectrochemical interest
practical applications???
mediated electron transfer
detection limit - 0.1 - 0.01 µM
direct electron transfer
detection limit - 5 - 10 µM
s u bs trate
s u bs trate
O2
oxidase
oxidase
produ ct
O2
H2 O2
HRP
H2 O
produ ct
red
e-
H2 O2
HRP
e le ctrode
H2 O
ox
s low proce s s
Peroxidase-modified electrodes: fundamentals and application.
Ruzgas, T; Csöregi, E; Emnéus, J; Gorton, L; Marko-Varga, G.
Anal. Chim. Acta (1996), 330(2-3)
red
ox
ox
me diator
e-
e le ctrode
red
very rapid proce s se s
Advantages with coimmobilising H2O2 producing oxidases with
peroxidases
 general approach for all H2O2 producing oxidases
 allows the oxidase to use its natural reoxidising agent (electron-proton acceptor),
molecular oxygen (O2)
 no competition between artificial mediator and O2
 some oxidases have no or very low reaction rates with artificial mediators
 allows the use of an applied potential within the "optimal potential range" (≈ -150
- +50 mV vs. SCE, pH 7)
 less interfering reactions from complex matrices
 electron transfer between electrode and peroxidase can be either direct or
mediated (control of response range and sensitivity)
Electron transfer in biosensors
 First generation
 Second generation
 Third generation
Mediators in bioelectrochemistry
1 e- acceptor/donors
vs.
2 e--H+ acceptor/
donors
hexacyanoferrate
Fe(CN)
methylene blue
4-/36
ferrocene
N
(H3C) 2 N+
S
N(CH 3 )2
N-methylphenazinium
0/1+
Fe
N
methylviologen
CH 3
N
•/+
N+
CH 3
anthraquinone
O
+
N
CH 3
O
1 e- acceptor/donor
2 e--H+ acceptor/donor
+E°’ does not vary with pH
no H+ participates
-E°’ varies with pH
1-2 H+ participate
+ no radical intermediates
stable redox reaction
-radical intermediates
unstable redox reaction
-low reaction rates with NADH
+ high reaction rates
with NADH
-moderate reaction rates with
peroxidases
+ high reaction rates
with peroxidases
overall redox chemistry
OH
O
+ 2H+ + 2 e-
OH
O
E°' will vary with pH
kET  e
-(G+ )2
- (d -d 0 )
4RT 
e
reaction in aqueous solutions occurs with intermediates

not redox stable
O
e-
H+
e-
H+
OH
i nte rme di ate
O
OH
1 electron non-proton acceptors/donors have been favoured
2+/3+
lately, e.g., ferrocenes, Os
-complexes
Marcus equation
The rate of electron transfer between two redox species is expressed by:
thermodynamic driving force
kET
-( G+ )2
-  (d -d 0 )
4RT

e
e
distance
reorganisation
energy
Examples of redox wires
2 ele ctron-proton acce ptor/donor
1 ele ctron acce ptor/donor
CH 3
1
N
12
N
( H2 C
CH) n
C O
NH 2
N
(H2 C
C) n
C O
CH 3
HN
S
H3 C
N
N
II/III
N
N Os
NN
Cl
A. Heller e t al.
Y. Okamoto e t al.
N+
CH 3
Example of an Os2+/3+-based redox polymer,
A. Heller, J. Phys. Chem., 96 (1992) 3579-3587
18
N
N
N
N
H 3C
N
Cl
Cl
Os II/III
N
N
N
CH 3
H 3C
CH3
PVI19-[Os(Me2-bpy)2Cl2]
formal potential (E°’) of mediator??????
mediators are ”general” electrocatalysts
new Os2+/3+-polymer, E°’ ≈ + 100 mV vs. Ag|AgCl
can it be further improved (i.e., lowered)?
for E°’-values below 0 mV: risk for electrocatalytic
reduction of O2
Which group(s) works best with mediators????
oxidase
dehydrogenase
S
O2
MEDox
S
MEDox
P
MEDred
H 2O 2
P
MEDred
peroxidase
NAD-dehydrogenase
AH
H2O2
S
NAD+
MEDred
P
NADH
MEDox
A•
AH
H2O
A•
Dehydrogenases with bound cofactors are the
”best” to wire because:
+ bound cofactor (c.f. NAD dehydrogenase)
+ not oxygen dependent ( c.f. oxidase)
but
- not so many (yet)
- often not so stable (c.f. GOx, HRP)
NAD-dependent dehydrogenase
+
S
NAD
MEDred
P
NADH
MEDox
Electrocatalytic oxidation of NAD(P)H on mediator- modified
electrodes.
Substrate + NAD(P)
+
dehydrogenase

Product + NAD(P)H + H+
obstacles to solve to make electrochemical sensors based on these enzymes:
1. both NAD(P)+ and NAD(P)H suffer from severe electrochemical irreversibility
2. enzyme depends on a soluble cofactor
3. the equilibrium of the reaction for most substrates favours the substrate NOT
the product side
NAD+ has a LOW oxidising power (E°'pH 7 = -560 mV vs. SCE)
Dehydrogenase with bound cofactor, e.g.,
glucose PQQ-dehydrogenase
S
MEDox
P
MEDred
L. Ye, M. Hämmerle, A. J. J. Olsthoorn, W. Schuhmann, H.-L. Schmidt, J.
A. Duine, A. Heller, High Current Density "Wired" Quinoprotein
Glucose Dehydrogenase Electrode
Anal. Chem., 65 (1993) 238-241
Engineered new enzymes tailormade for
biosensor applications
i GDH-PQQ membrane bound enzyme
i PQQ loosely bound to the enzyme
i Different GDH-PQQ have different selectivities
i Different GDH-PQQ have different pH optima
=> through genetic engineering combine the ”best”
properties of each of several GDH-PQQs and produce a
new ”optimal” glucose oxidising enzyme
Bioengineered (new) enzymes
Construction of multi-chimeric pyrroloquinoline quinone
glucose dehydrogenase with improved enzymatic
properties and application in glucose monitoring.
Yoshida, H; Iguchi, T; Sode, K. Biotechnology Letters
(2000), 22(18), 1505-1510.
Secretion of water soluble pyrroloquinoline quinone
glucose dehydrogenase by recombinant Pichia pastoris.
Yoshida, H; Araki, N; Tomisaka, A; Sode, K. Enzyme
Microb. Technol. (2002), 30(3), 312-318.
New electrode materials
Walcarius, Alain. Electrochemical Applications of Silica-Based OrganicInorganic Hybrid Materials. Chemistry of Materials (2001), 13(10),
3351-3372
Walcarius, Alain. Electroanalysis with pure, chemically modified, and
sol-gel-derived silica-based materials. Electroanalysis (2001), 13(8-9),
701-718
Walcarius, Alain. Zeolite-modified electrodes in electroanalytical
chemistry. Anal. Chim. Acta (1999), 384(1), 1-16.
Walcarius, Alain. Analytical applications of silica-modified electrodes. A
comprehensive review. Electroanalysis
(1998),10(18), 1217-1235
Electron transfer in biosensors
 First generation
 Second generation
 Third generation
schematic picture of a redox enzyme on an electrode surface
active site
e-
electrode
efficient direct electron
transfer has been shown for
some redox enzymes mainly
those containing
i: heme
ii: iron-sulphur clusters
iii: copper
Table 1. Redox enzymes for which DET reactions with electrodes have been shown, adapted and updated after [19].
enzyme
laccases
Polyporus versicolor
Rhus vernicifera
Coriolus hirsitus
cofactor
4 Cu
4 Cu
4 Cu
4 Cu
substrate
O2
O2
O2
O2
redox reaction
reduction
reduction
reduction
reduction
reference
ascorbate oxidase
4 Cu
O2
reduction
[30]
superoxide dismutase
Cu-Zn
O2 •
peroxidases
heme
H 2 O2
horseradish
soybean peroxidase
tobacco peroxidase
sweet potato peroxidase
peanut peroxidase
fungal peroxidase
cytochrome c peroxidase
chloroperoxidase
cytochrome c peroxidase
Paracoccus denitrificans
bovine lactoperoxidase
microperoxidase
hydrogenase
heme
heme
heme
heme
heme
heme
heme
heme
2 hemes
[4,33]
[34]
[34,35]
heme
heme
Fe-S cluster
[45,46]
[45-48]
[49]
methylamine dehydrogenase
diaphorase
Bacillus stearothermophilus
bi-functional enzymes
cytochrome b2
lactate dehydrogenase
p-cresolmethylhydrolase
flavocytochrome c552
cellobiose dehydrogenase
Phanerochaete chrysosporium
Sclerotium rolfsii
bi-functional enzymes
D-fr uctose dehydrogenase
alcohol dehydrogenase
Gluconobacter suboxydans
Acetobacter aceti
Gluconobacter oxydans
bi-functional enzymes
succinate dehydrogenase
fumarate reductase
trifunctional enzymes
D-gluconate dehydrogenase
FAD-Fe-S cluster
flavo-heme-Fe-S cluster
FAD-heme-Fe-S cluster
[3]
[29]
[29]
[31]
reduction
[32]
[35]
[36,37]
[38-42]
[43]
[44]
oxidation
reduction
methoxatin-like quinone
FAD
H2
+
H
methylamine
NADH
oxidation
oxidation
[50]
[51,52]
flavo-heme
FMN-heme
lactate
oxidation
[53]
p-cresol
sulfide
cellobiose,
lactose
cellodextrins
oxidation
oxidation
oxidation
[54]
[55]
fructose
ethanol
oxidation
oxidation
FAD-heme
FAD-2 heme
FAD-heme
FAD-heme
PQQ-heme
PQQ-heme
PQQ-4 hemes
PQQ-4 hemes
PQQ-4 hemes
FAD Fe-S cluster
FAD Fe-S cluster
[56,57]
[58-61]
[51,62,63]
[64]
[65]
succinate
fumarate
fumarate
oxidation
reduction
reduction
[66,67]
[68]
D-gluconate
oxidation
[51,69,70]
Gazarya n, Anal. Chim. Acta., 400 (1999) 91-108
.
L. Gorton, A. Lindgren, T. Larsson, F. D. Munteanu, T. Ruzgas and I.
L.-H. Guo and H. A. O. Hill, Adv. Inorg. Chem., 36 (1991) 341-373
“There appear to be two classes of redox enzymes: intrinsic
and extrinsic”
Intrins ic:
Extrins ic
Catalytic reaction between an enzyme
and its substrate takes place within a
highly localised assembly of redoxactive sites. There need be no electron
transfer pathways from these sites to the
surface of the enzyme, where, it is
presumed, it would interact with an
electrode. For such intrinsic redox
enzymes, electrode reactions may
require:
(1) that the sites of the catalytic reaction
be close to the protein surface
(2) that the enzyme can deform without
loss of activity
(3) that the electrode surface projects
into the enzyme
(4) that electron pathways be introduced
by modification of the enzyme
With extrinsic redox enzymes, there is
usually another protein involved in
transporting electrons and therefore an
electron transfer pathway exists within
the enzyme connecting the active sites to
an area on the surface where the
ancillary protein binds. If this area
could be disposed toward an electrode,
it would be possible for the enzyme
electrochemistry to be obtained.
intrinsic enzyme
insulating protein shell
extrinsic enzyme
built in ET pathways
Random adsorption/orientation on
carbon
< 100% of enzyme molecules in direct ET
contact with the electrode
ordered orientation on thiol modified gold
high % (≈ 100%) of enzyme molecules in DET contact with the electrode
Self-assembled monolayers as an
orientation tool - Reconstitution
+ apo-HRP
+ hemin (and EDC)
+ diaminoalkane
mixed SAM
e.g., GOx, GDH-PQQ
Gold
H. Zimmermann, A. Lindgren, W. Schuhmann, L. Gorton, Chem. Eur. J. 6 (2000) 592-599
Peroxidase
• Peroxidases are
found in
–
–
–
–
Structure of horseradish
peroxidase (HRP) C
Plants
Bacteria
Fungi
Animal tissues
• Cofactor  heme
M. Gajhede, et.al., Nature Structural Biology, 4 (1997) 1032.
Structural Models of Recombinant (left) and
Native Glycosylated (right) Horseradish
Peroxidase C
Hydrophobic residues are coloured in red and hydrophilic in blue
Structural Model of Recombinant Horseradish
Peroxidase C with a His-tag located at either the
C- or the N-terminus
ket and % in DET between HRP and electrode
H2 O2
H2 O
Native POD
electrode
k1
k et
2e H2 O
Compound-I
0 mV vs.
Ag/AgCl
2H+
native HRP/graphite ≈ 2 s-1 (50% DET)
rec HRP/graphite ≈ 8 s-1 (65%)
rec HRP/gold ≈ 18 s-1 (60%)
CHisrec HRP/gold ≈ 35 s-1 (75%)
NHisrec HRP/gold ≈ 30 s-1 (65%)
Direct electron transfer
• In the presence of
enzyme substrate
• In the absence of
enzyme substrate
Direct electron transfer of CDH
• Cyclic voltammetry of CDH • Electrocatalytic current
100 mV/s
50 mV/s
20 mV/s
10 mV/s
1
+ 3.8 mM cellobiose
0.8
Current/µA
Current/µA
Current/µA
Current/µA
0.5
0
0.6
0.4
0.2
0
E°’=-41±3 mV
-0.5
-250 -200 -150 -100 -50
0
50 100 150
Potential/mV vs Ag/AgCl
CDH trapped under a membrane at a
gold electrode (modified with cystamine)
in 50 mM Ac-buffer, pH 5.1.
-0.2
-0.4
-200
-100
0
100
200
300
Potential/mV vs Ag/AgCl
pH 4.4, scanrate 50 mV/s
A. Lindgren, T. Larsson, T. Ruzgas, L. Gorton, J. Electroanal. Chem., 494 (2000) 105-113
400
Electrocatalysis at the CDH electrode
0.8
Current/µA
Current/µA
• Electrocatalytic current
was observed in the
presence of the enzyme
substrate, cellobiose.
1
pH 3.6
pH 4.4
pH 5.1
pH 6.0
0.6
0.4
0.2
0
• At high pH the internal
ET is decreased
-0.2
-0.4
1
• Low pH
FAD
Heme
Current/µA
Current/µA
0.8
0.6
0.4
0.2
0
• High pH
-0.2
-0.4
-200
FAD
Heme
-100
0
100
200
300
Potential/mV vs Ag/AgCl
400-200
-100
0
100
200
300
Potential/mV vs Ag/AgCl
With 3.8 mM cellobiose, without cellobiose
50 mM Ac-buffer, scan rate 50 mV s-1.
400