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Communication betweenredoxenzyme and electrode
active site
electrode
wanted electron transfer reactione-
substrate
product
oxidised enzyme
reduced enzyme
electron transfer electron transfer
electrode
2 e-
Wanted electron transfer path for fundamental studies and practical applications e.g., in biosensors (here examplified with an oxidation reaction)
fundamental bio-electrochemical interest as direct electron transfer reactions between oxidoreductases and electrodes are seldom reported
active site
electrode
=direct electron transfer blocked due to steric or kinetic restrictionse-
Electron transfer in biosensors
Substrate Product
Substrate Product
Substrate Product
O2 H2O2 Medox Medred
First generation Second generation Third generation
active site
electrode
electron transfer occurs in two steps: 1: between enzyme and mediator2: between mediator and electrode
e-
facilitate electron transfer between the redox enzyme and the electrode with a mediator
e-
enzyme
mediator
Mediated electron transfer path between a redox enzyme and an electrode
reduced mediator
oxidised mediator
electrode
2 e-electron transfer
substrate
product
oxidised enzyme
reduced enzyme
electron transfer electron transfer
Major groups of redox enzymes used in biosensor work
S S
S
PP
P
NAD+
NADH MEDox
MEDred
MEDoxO2
H2O2
MEDox
MEDredMEDred
H2O2
H2O
AH
AH
A•
A•
oxidase dehydrogenase
NAD-dehydrogenase peroxidase
+600+500+400+300+200+100-700 -600 -500 -400 -300 -200 -100E/mV vs. SCE at pH 7.0
0
peroxidases (haeme) compound I/II
NAD+/NADH -560 mV
glucose/gluconolactone -635 mV
Optimal potential range
enzyme bound flavins/PQQ haeme (Fe2+/3+)
E / mV vs. Ag/AgCl-300 -200 -100 0 +100
optimal potential range -200 and 0 mV vs. Ag/AgCl (at pH 7. 0)
* low background current, low noise
* no O2 reduction
* no (or very small) oxidation of ascorbic acid uric acid paracetamol etc.
Electron transfer in biosensors
First generation Second generation Third generation
Substrate Product
Substrate Product
Substrate Product
O2 H2O2 Medox Medred
First generation biosensors
substrate
oxidase
product
O2
H2O2
2 H+ + O2
electrode
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
noble metal deposition on carbon electrodes
Prussian Blue deposition on conventional electrodes
peroxidase modified electrodes
other catalysts e.g. iron phthalocyanine
noble metal (Pt, Pd, Ru, Rh) deposition on carbon electrodes
lack of selectivity - future????
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.
substrate
oxidase
product
O2
H2O2
2 H2O
electrode
2 e-
2 H+
substrate
oxidase
product
O2
H2O2
2 H+ + O2
electrode
2 e-
oxidation at +200-300 mV vs. Ag|AgCl reduction at 0 - -150 mV vs. Ag|AgCl
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
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
substrate
oxidase
product
O2
H2O2
2 H2O
electrode
2 e-
2 H+
reduction at +150 - -150 mV vs. Ag|AgCl
peroxidase modified electrodes
of great bioelectrochemical interest
practical applications???
Peroxidase-modified electrodes: fundamentals and application.Ruzgas, T; Csöregi, E; Emnéus, J; Gorton, L; Marko-Varga, G. Anal. Chim. Acta (1996), 330(2-3)
substrate
oxidase
product
O2
H2O2
H2O
red
ox
HRP electrodee-
slow process
direct electron transfer detection limit 5 - 10 µM
ox
red
mediator electrodee-
very rapid processes
mediated electron transfer detection limit 0.1 - 0.01 µM
substrate
oxidase
product
O2
H2O2
H2O
red
ox
HRP
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
Substrate Product
Substrate Product
Substrate Product
O2 H2O2 Medox Medred
Mediators in bioelectrochemistry1 e- acceptor/donors vs. 2 e--H+ acceptor/
donors
N
N
CH3
CH3
S
N
(H3C)2N+ N(CH3)2
N+
N
CH3
O
O
Fe(CN)64-/3-
Fe0/1+
hexacyanoferrate
ferrocene
methylviologen
methylene blue
•/+
+
N-methylphenazinium
anthraquinone
1 e- acceptor/donor 2 e--H+ acceptor/donor
+E°’ does not vary with pH -E°’ varies with pHno H+ participates 1-2 H+ participate
+ no radical intermediates -radical intermediatesstable redox reaction unstable redox reaction
-low reaction rates with NADH + high reaction rates with NADH
-moderate reaction rates with + high reaction rates peroxidases with peroxidases
kET e-(d -d0 )
e
-(G + )2
4RT
O
O
OH
OH
O
O
OH
OH
+ 2H+ + 2 e-
overall redox chemistry
E°' will vary with pH
e-
intermediate
H+ e- H+
reaction in aqueous solutions occurs with intermediates not redox stable
1 electron non-proton acceptors/donors have been favoured lately, e.g., ferrocenes, Os 2+/3+-complexes
Marcus equation
The rate of electron transfer between two redox species is expressed by:
kET e-(d-d0)
e-(G+ )2
4RT
distance
thermodynamic driving force
reorganisationenergy
N
S N+CH3
CH3
HN
C O
C)n
CH3
H2CCH)n
C
NH2
O
H2C ((
H3C
Examples of redox wires
Os
N
N
N
N
N
N
N
N
Cl
II/III
121
1 electron acceptor/donor 2 electron-proton acceptor/donor
A. Heller et al. Y. Okamoto et al.
Example of an Os2+/3+-based redox polymer, A. Heller, J. Phys. Chem., 96 (1992) 3579-3587
H3C
H3C
CH3
CH3
N
N
N
N
Os
Cl
N
N
N
N
18
II/IIICl
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????
S S
S
PP
P
NAD+
NADH MEDox
MEDred
MEDoxO2
H2O2
MEDox
MEDredMEDred
H2O2
H2O
AH
AH
A•
A•
oxidase dehydrogenase
NAD-dehydrogenase peroxidase
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)
Electrocatalytic oxidation of NAD(P)H on mediator- modified electrodes.
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 sideNAD+ has a LOW oxidising power (E°'pH 7 = -560 mV vs. SCE)
Substrate + NAD(P)+ dehydrogenase
Product + NAD(P)H + H+
Dehydrogenase with bound cofactor, e.g., glucose PQQ-dehydrogenase
S
P
MEDox
MEDredL. Ye, M. Hämmerle, A. J. J. Olsthoorn, W. Schuhmann, H.-L. Schmidt, J.A. Duine, A. Heller, High Current Density "Wired" QuinoproteinGlucose Dehydrogenase ElectrodeAnal. Chem., 65 (1993) 238-241
Engineered new enzymes tailormade for biosensor applications
GDH-PQQ membrane bound enzyme
PQQ loosely bound to the enzyme
Different GDH-PQQ have different selectivities
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 materialsWalcarius, Alain. Electrochemical Applications of Silica-Based Organic-Inorganic 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
Substrate Product
Substrate Product
Substrate Product
O2 H2O2 Medox Medred
active site
electrode
efficient direct electron transfer has been shown for some redox enzymes mainly those containing i: hemeii: iron-sulphur clusters iii: copper
e-
schematic picture of a redox enzyme on an electrode surface
Table 1. Redox enzymes for which DET reactions with electrodes have been shown, adapted and updated after [19].
enzyme cofactor substrate redox reaction referencelaccases 4 Cu O2 reduction
Polyporus versicolor 4 Cu O2 reduction [3]
Rhus vernicifera 4 Cu O2 reduction [29]
Coriolus hirsitus 4 Cu O2 reduction [29]
ascorbate oxidase 4 Cu O2 reduction [30]
superoxide dismutase Cu-Zn O2• [31]
peroxidases heme H2O2 reduction [32]
horseradish heme [4,33]soybean peroxidase heme [34]tobacco peroxidase heme [34,35]sweet potato peroxidase hemepeanut peroxidase heme [35]fungal peroxidase heme [36,37]cytochrome c peroxidase heme [38-42]chloroperoxidase heme [43]cytochrome c peroxidaseParacoccus denitrificans
2 hemes [44]
bovine lactoperoxidase heme [45,46] microperoxidase heme [45-48]hydrogenase Fe-S cluster H2
H+
oxidationreduction
[49]
methylamine dehydrogenase methoxatin-like quinone methylamine oxidation [50]diaphoraseBacillus stearothermophilus
FAD NADH oxidation [51,52]
bi-functional enzymes flavo-hemecytochrome b2lactate dehydrogenase
FMN-heme lactate oxidation [53]
p-cresolmethylhydrolase FAD-heme p-cresol oxidation [54]flavocytochrome c552 FAD-2 heme sulfide oxidation [55]
cellobiose dehydrogenase Phanerochaete chrysosporium Sclerotium rolfsii
FAD-hemeFAD-heme
cellobiose,lactosecellodextrins
oxidation[56,57]
bi-functional enzymes PQQ-hemeD-fructose dehydrogenase PQQ-heme fructose oxidation [58-61]alcohol dehydrogenaseGluconobacter suboxydansAcetobacter acetiGluconobacter oxydans
PQQ-4 hemesPQQ-4 hemesPQQ-4 hemes
ethanol oxidation[51,62,63][64][65]
bi-functional enzymes FAD Fe-S clustersuccinate dehydrogenase FAD Fe-S cluster succinate
fumarateoxidationreduction
[66,67]
fumarate reductase FAD-Fe-S cluster fumarate reduction [68]trifunctional enzymes flavo-heme-Fe-S clusterD-gluconate dehydrogenase FAD-heme-Fe-S cluster D-gluconate oxidation [51,69,70]
L. Gorton, A. Lindgren, T. Larsson, F. D. Munteanu, T. Ruzgas and I.Gazaryan, Anal. Chim. Acta., 400 (1999) 91-108.
L.-H. Guo and H. A. O. Hill, Adv. Inorg. Chem., 36 (1991) 341-373
“There appear to be two classes of redox enzymes: intrinsicand extrinsic”
Intrins ic:
Catalytic reaction between an enzymeand its substrate takes place within ahighly localised assembly of redox-active sites. There need be no electrontransfer pathways from these sites to thesurface of the enzyme, where, it ispresumed, it would interact with anelectrode. For such intrinsic redoxenzymes, electrode reactions mayrequire:(1) that the sites of the catalytic reactionbe close to the protein surface(2) that the enzyme can deform withoutloss of activity(3) that the electrode surface projectsinto the enzyme(4) that electron pathways be introducedby modification of the enzyme
Extrins ic
With extrinsic redox enzymes, there isusually another protein involved intransporting electrons and therefore anelectron transfer pathway exists withinthe enzyme connecting the active sites toan area on the surface where theancillary protein binds. If this areacould be disposed toward an electrode,it would be possible for the enzymeelectrochemistry to be obtained.
Random adsorption/orientation on carbon
< 100% of enzyme molecules in direct ET contact with the electrode
O OH OO
OHOOH O OH O
OOHO
OH O OH O
OOHO
OH O OH O
OOHO
OH
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
Gold
mixed SAM
+ diaminoalkane
+ hemin (and EDC)
+ apo-HRP
e.g., GOx, GDH-PQQ
H. Zimmermann, A. Lindgren, W. Schuhmann, L. Gorton, Chem. Eur. J. 6 (2000) 592-599
• Peroxidases are found in– Plants– Bacteria– Fungi– Animal tissues
• Cofactor heme
Structure of horseradish peroxidase (HRP) C
M. Gajhede, et.al., Nature Structural Biology, 4 (1997) 1032.
Peroxidase
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
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%)
Native POD
Compound-I2H+
H2O
H2O
H2O2
k1
2e-
electrode
0 mV vs.Ag/AgCl
ket
Direct electron transfer
• In the presence of enzyme substrate
• In the absence of enzyme substrate
Substrate Product
Direct electron transfer of CDH• Electrocatalytic current• Cyclic voltammetry of CDH
CDH trapped under a membrane at a gold electrode (modified with cystamine) in 50 mM Ac-buffer, pH 5.1.
-0.5
0
0.5
-250 -200 -150 -100 -50 0 50 100 150
100 mV/s50 mV/s20 mV/s10 mV/s
Cu
rre
nt/
µA
Potential/mV vs Ag/AgCl
Cu
rren
t/µ
A
E°’=-41±3 mV
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
-200 -100 0 100 200 300 400
Cu
rre
nt/
µA
Potential/mV vs Ag/AgClC
urr
ent/
µA
+ 3.8 mM cellobiose
pH 4.4, scanrate 50 mV/s
A. Lindgren, T. Larsson, T. Ruzgas, L. Gorton, J. Electroanal. Chem., 494 (2000) 105-113
Electrocatalysis at the CDH electrode• Electrocatalytic current was
observed in the presence of the enzyme substrate, cellobiose.
• At high pH the internal ET is decreased
• Low pH
• High pH
With 3.8 mM cellobiose, without cellobiose 50 mM Ac-buffer, scan rate 50 mV s-1.
-200 -100 0 100 200 300 400
Potential/mV vs Ag/AgCl
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
Cu
rre
nt/
µA
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
-200 -100 0 100 200 300 400
Cu
rre
nt/
µA
Potential/mV vs Ag/AgCl
pH 3.6 pH 4.4
pH 5.1 pH 6.0
FAD Heme
FAD Heme
Cu
rre
nt/
µA
Cu
rre
nt/
µA
Recommended