Electrochemistry of various cellobiose dehydrogenases

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Communication betweenredoxenzyme and electrode
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Electron transfer in biosensors

• First generation

• Second generation

• Third generation

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Major groups of redox enzymes used in biosensor
work
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Electron transfer in biosensors

• First generation

• Second generation

• Third generation

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• First generation biosensors

at conventional electrodes electrochemical
oxidation of H2O2 occurs at 600 mV vs.
AgAgCl the system is open for interfering
reactions the response is unstable with time
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Ways to reduce the potential for electrochemical
conversion of H2O2i noble metal deposition on
carbon electrodesi Prussian Blue deposition on
conventional electrodesi peroxidase modified
electrodesi other catalysts e.g. iron
phthalocyanine
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noble metal (Pt, Pd, Ru, Rh) deposition on carbon
electrodeslack 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),
247-53Catalytic 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), 3-7Electrochemical metalization of
carbon electrodes. O'Connell, P. J. O'Sullivan,
C. K. Guilbault, G. G. Anal. Chim. Acta
(1998), 373(2-3), 261-270.
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deposition of Prussian Blue and related
catalysts on conventional electrodes selective
electroreduction of H2O2 at around 0 mV vs.
AgAgCl- lack of long term stability at pH gt
7.5 Prussian Blue and its
analogues electrochemistry and analytical
applications. Karyakin, A. A.. Electroanalysis
(2001), 13(10), 813-819Metal-hexacyanoferrate
films A tool in analytical chemistry. de
Mattos, Ivanildo Luiz Gorton, Lo. Quimica Nova
(2001), 24(2), 200-205
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peroxidase modified electrodesof great
bioelectrochemical interestpractical
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)
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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)
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Electron transfer in biosensors

• First generation

• Second generation

• Third generation

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Mediators in bioelectrochemistry1 e-
acceptor/donors vs. 2 e--H acceptor/
donors
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1 e- acceptor/donor 2 e--H
acceptor/donorE does not vary with pH -E
varies with pH no H participates 1-2 H
participate no radical intermediates -radical
intermediates stable 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
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Marcus equation
The rate of electron transfer between two redox
species is expressed by
thermodynamic driving force
reorganisation energy
distance
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Example of an Os2/3-based redox polymer, A.
Heller, J. Phys. Chem., 96 (1992) 3579-3587
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formal potential (E) of mediator??????mediator
s are general electrocatalystsnew
Os2/3-polymer, E 100 mV vs. AgAgCl can
it be further improved (i.e., lowered)?for
E-values below 0 mV risk for electrocatalytic
reduction of O2
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Which group(s) works best with mediators????
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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)
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NAD-dependent dehydrogenase
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Electrocatalytic oxidation of NAD(P)H on
mediator- modified electrodes.obstacles to
solve to make electrochemical sensors based on
these enzymes1. both NAD(P) and NAD(P)H
suffer from severe electrochemical
irreversibility2. enzyme depends on a soluble
cofactor3. 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)
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Dehydrogenase with bound cofactor, e.g., glucose
PQQ-dehydrogenase
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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

gt through genetic engineering combine the best
properties of each of several GDH-PQQs and
produce a new optimal glucose oxidising enzyme
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Bioengineered (new) enzymesConstruction 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.
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New electrode materials

• Walcarius, 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

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Electron transfer in biosensors

• First generation

• Second generation

• Third generation

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L.-H. Guo and H. A. O. Hill, Adv. Inorg. Chem.,
36 (1991) 341-373
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Random adsorption/orientation on carbonlt 100
of enzyme molecules in direct ET contact with the
electrode
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ordered orientation on thiol modified goldhigh
( 100) of enzyme molecules in DET contact
with the electrode
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Self-assembled monolayers as an orientation tool
- Reconstitution
e.g., GOx, GDH-PQQ
H. Zimmermann, A. Lindgren, W. Schuhmann, L.
Gorton, Chem. Eur. J. 6 (2000) 592-599
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Peroxidase
Structure of horseradish peroxidase (HRP) C

• Peroxidases are found in
• Plants
• Bacteria
• Fungi
• Animal tissues
• Cofactor ? heme

M. Gajhede, et.al., Nature Structural Biology, 4
(1997) 1032.
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Structural Models of Recombinant (left) and
Native Glycosylated (right) Horseradish
Peroxidase C
Hydrophobic residues are coloured in red and
hydrophilic in blue
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Structural Model of Recombinant Horseradish
Peroxidase C with a His-tag located at either the
C- or the N-terminus
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ket and in DET between HRP and
electrodenative 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)
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Direct electron transfer

• In the presence of enzyme substrate

• In the absence of enzyme substrate

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Direct electron transfer of CDH

• Electrocatalytic current

• Cyclic voltammetry of CDH

Current/µA
E-413 mV
CDH trapped under a membrane at a gold electrode
(modified with cystamine) in 50 mM Ac-buffer, pH
5.1.
A. Lindgren, T. Larsson, T. Ruzgas, L. Gorton,
J. Electroanal. Chem., 494 (2000) 105-113
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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

pH 3.6
pH 4.4
Current/µA
pH 5.1
pH 6.0
Current/µA
With 3.8 mM cellobiose, without cellobiose 50 mM
Ac-buffer, scan rate 50 mV s-1.