TOPICS IN (NANO) BIOTECHNOLOGY

Enzyme sensors

30th June

PhD Course

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)

NAD-dependent dehydrogenase

S

P

NAD+

NADH MEDox

MEDred

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.

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

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