By Ivo Frébort Biosensors A focus on peroxidase-modified electrodes and their practical applications.

by

Ivo Frébort

Biosensors

A focus on peroxidase-modified electrodes and their practical applications

Consists of: biocatalyst (enzyme, cells, tissue)

transducer (converts the biological or biochemical signal into a quantifiable electrical or optical signal)

Biosensor- an analytical device that exploits a biocatalytic reaction

Leland C. Clark, Jr. with the first enzyme electrode

Oxygen electrode (1956)

working electrode: Pt cathode (-0.6 V) reference electrode: Ag/AgCl

electrodes separated from measured solution with a gas permeable mebrane

First biosensor - Clark (1962): glucose sensor with glucose oxidase and oxygen electrode

Glucose + O2 Gluconic acid + H2O2

electrodeo-ring

E E E E E dialyzing

membrane

O CH (CH2)3 CH O

CH O H2N

CH N

CH2 NH

Glutaraldehyde

+

Schiff base

Reduction with NaBH4

E E

E

E

BSA

BSABSA

BSABSA

Construction of the biosensors

Sensing electrode: platinum, gold, various forms of carbon

Immobilization techniques: general method doesn’t exist- enzyme physical entrapment- covalent crosslinking

BB

NH2

O CH (CH2)3 CH ON CH (CH2)3 CH O

B

NH CH (CH2)3 CH NHN CH (CH2)3 CH N

NH2

NaBH4

Glutaraldehyde reaction

COOH +

N

C

N

R

R'

CO

O C

N+

NH

R'

R

CO

NH B

NH2B

Carbodiimide reaction

C O

NH

NH R'

R

N C N

CH3CH2 N C N (CH2)3 N(CH3)2

N C N CH2CH2 N O

H3C

DCC

CMC

tosyl-

EDC

Covalent attachment to a support membrane or the electrode

S (CH2)2

S (CH2)2

NH2

NH2Au

S (CH2)2 NH2

S (CH2)2 NH2Au

+

X

X

SSSSSS

XXXXXX

SSSSSS

Organized layer Dilution with an inert thiol

R Si(OC2H5)3 O Si R

O

O

Si R

O

O

(C2H5)3Si (CH2)3 NH2

(CH3)3Si (CH2)3 O CH2 CH CH2O

Si

CH3

(CH2)3

CH3

C2H5O NH2

--OH

--OH

APTES

GOPS

APDMES

Adsorption - glass, carbon, Au, Pt

- often activation needed

Adsorption of thiols to a gold electrode Silanization of an oxidized metal electrode

Screen print

Matrix carrier

Mobile wiper

Paste

Screen grid

Screen printing

3. Detection: Steady-state or flow injection analysis

An enzyme electrode

1. Thin enzyme layer with high specific activity, 2. Good selection of membranes

Response controlled by diffusion through the permselective membrane (not by enzyme kinetics)Enzyme activity low - thick membrane needed to achieve linear response, response slowEnzyme activity high - thin membrane OK, rapid response

Protective

membraneEnzyme layer Permselective

membrane

ElectrodeSample E

P1 P1

S1

S2

P2 P2

P2*

-

-

-

-

I

Background noise

Analyte Signal

S

dS/dt

Time

S

c

S/c Linear response

Detectionlimit

Biosensor parameters1. Sensitivity2. Linear response3. Detection limit4. Background noise5. Baseline drift6. Selectivity7. Response8. Operating stability9. Shelf life

S = 3 NN

Analyte

Assay of the detection limit

Type of measurement

A

BTime

S

Přímý kontakt se vzorkemDirect contact with the sampleDirect contact with the sample

S

Time

Analyte additions

Solution placed in a chamberSolution placed in a chamber

Flow cellFlow cell

IN OUT

Analyte Enzyme Reaction

Alcohol Alcohol oxidase Ethanol + O2 Acetaldehyde + H2O2

D-Glucose Glucose oxidase β-D-Glucose + O2 Gluconic acid + H2O2

Lactose Galactose oxidase Lactose + O2 Galactose dialdehyde der. + H2O2

L-Lactate L-Lactate oxidase L-Lactate + O2 Pyruvate + H2O2

Starch Amyloglucosidase Starch + H2O β-D-Glucose Glucose oxidase β-D-Glucose + O2 Gluconic acid + H2O2

Sucrose Invertase Sucrose + H2O α-D-Glucose + β-D-Fructose Mutarotase α-D-Glucose β-D-Glucose Glucose oxidase β-D-Glucose + O2 Gluconic acid + H2O2

First generation biosensors - response to the substrates in solution

1. Reduction of oxygen with a Clark type electrode at -0.6 V (vs SCE)2. Oxidation of hydrogen peroxide at a Pt electrode at +0.7 V 3. Measuring of pH change

Glucose + O2 Gluconic acid + H2O2

Examples of hydrogen peroxide measurning biosensor

Types of transducers used in biosensors

Type Detectable speciesAmperometric O2, H2O2, I2, NADH

Ion-selective electrode H+, Na+, Cl-

Field effect transistors H+, Na+, Cl-

Gas sensing electrode CO2, NH3

Photomultiplier Light emission ATP/Luciferase/Luciferin, H2O2/Peroxidase/Luminol, etc.

Thermistor Heat of reaction

Second generation biosensors- mediated electron transfer between enzyme and electrode- can be easily miniaturizedblood glucose measuring system in situ

Third generation biosensors- direct electron transfer between enzyme and electrode

Cell-based based biosensors- cheaper than purified enzymes,Nocardia erythropolis cells immobilised in polyacrylamide or agar(cholesterol oxidase)Cholesterol + O2 Cholest-4-en-3-one + H2O2

Enzyme immunosensors- many types, based on ELISA techniques- often use chemiluminiscence or bioluminiscencehuman chorionic gonadotropin - pregnancy

Examples of biosensors

Analyte Biocatalyst Transducer Immobilization Shelf life Response

Alcohol Alcohol oxidase O2 Glutaraldehyde 2 weeks 1-2 minArginine Streptococcus faecium NH3 Entrapment 3 weeks 20 minCholesterol Nocardia erythropolis O2 Entrapment 4 weeks 35-70 sD-Glucose Glucose oxidase O2 Covalent 3 weeks 1 minGlutamate Glutamate decarboxylase CO2 Glutaraldehyde 1 week 10 minNAD+ NADase + Escherichia coli NH3 Membrane 1 week 5-10 minNitrate Azotobacter vinelandii NH3 Entrapment 2 weeks 7-8 minPenicillin Penicillinase H+ Polyacrylamide 2 weeks 15-30 sUrea Urease NH4

+ Polyacrylamide 3 weeks 20-40 s

Personal glucose meter for diabetics (Medisense Britain, Ltd.)

Biacore 2000 (Biacore)www.bioacore.com

IAsys (Affinity Sensors)www.affinity-sensors.com

KI 1 (BioTuL)www.biotul.com

IBIS II (XanTec)www.xantec.com

Automated affinity systems

Peroxidase-based electrodes

Ruiz-Duenas, F. J., Martinez, M. J., Martinez, A. T.: Peroxidase from the ligninolytic fungus Pleurotus eryngii. Mol Microbiol 31 pp. 223 (1999)

Protein of 35-45 kDa, prosthetic group - heme, Mn2+

Convenient sources: horse radish root, soybean, tobacco leaves, various fungi

PEROXIDASE (EC 1.11.1.7)

Native peroxidase + H2O2

(Fe3+)Compound-I + H2O(Fe4+=O, Por+)

Compound-I + AH2

(Fe4+=O, Por+)Compound-II + AH*(Fe4+=O)

Compound-II + AH2

(Fe4+=O)Native peroxidase+ AH* + H2O(Fe3+)

The catalytic cycle of peroxidase

Applications of peroxidase-based electrodes

1. Detection of hydrogen peroxide in aqueous solutions industry, environmental protection, clinical control photochemical smog, pathological processes in lungs, etc.

2. Detection of organic peroxides in water and organic solutions free radical injury, oxidative stress, autooxidation of unsaturated lipids

3. Detection of compounds based on peroxidase inhibition CN-, F-, hydroxylamine

4. Detection of aromatic amines and phenolic compounds environmental control: chlorophenols in water

5. Immunosensors based on peroxidase electrodes peroxidase conjugates with antibody, H2O2-producing enzyme conjugates

6. Detection of analytes based on peroxidase/oxidase-coupled reactions glucose, ethanol, lactate,choline, xanthine, cholesterol, bilirubin, glutamate

A. Surface modified electrodes Electrode material: graphite, glassy carbon, gold, SnO2

Coupling: carbodiimide, glutaraldehyde, adsorption

B. Polymer-based electrodes Crosslinking with Os(bpy)2

3+/2+ redox polymer, electropolymerized polypyrrole, o-phenylethylamine

C. Bulk modified composite electrodes Graphite-silicone oil paste, paraffin oil paste, epoxy composite

D. Tissue-modified carbon paste electrodes Asparagus tissue, tobacco callus tissue, horseradish root, kohlrabi skin

Electrode designs

H2O

H2O2

2H+

H2O

e-

e-

Electrode

Eappl< 0.6 V

vs SCE

Compound-I(Fe4+=O, Por+)

Compound-II(Fe4+=O)

Ferriperoxidase(Fe3+)

Mechanism of direct biocatalytic reduction of hydrogen peroxide at peroxidase-modified electrodes

H2O

H2O2

2H+

H2O

Electrode

Compound-I(Fe4+=O, P+)

Compound-II(Fe4+=O)

Ferriperoxidase(Fe3+)

Mred

Mox

Mred

Mox

Mechanism of mediated biocatalytic reduction of hydrogen peroxide at peroxidase-modified electrodes

Mediator: ferrocene, o-phenylenediamine, hydroquinone

The mediators

Ferrocene o-Phenylenediamine Hydroquinone

Fe+

R

NH2

NH2

OH

OH

Detailed look at a practical example ...

Copper amine oxidase-based electrodes for the assay of biogenic amines

Monitoring the biomarkers of food freshness: histamine, putrescine, cadaverineCurrently used methods: chromatographic techniques - they often require sample pre-treatment steps and skilled operators; the relatively long analysis time and high costs make these methods not suitable for routine useAim of the work: design and construction of the amperometric biosensors for monitoring of biomarkersTwo biosensor designs: monoenzymatic and bienzymatic, using both the direct and mediated electron transfer pathwaysBiological recognition element: copper amine oxidase (EC 1.4.3.6)Mediator: poly(1-vinylimidazole) complexed with [Os(4,4'-dimethylbipyridine)2Cl]+/2+ (PVI13-dmeOs)

Assay system: The biosensors were used in a flow-injection analysis (FIA) line

The biogenic amines: histamine, putrescine and cadaverine

H2N-(CH2)n-NH2

n=4: Putrescine; n=5: Cadaverine

Formed by the biodegradation of the aminoacids ornithine and lysine by the action of putrefactive bacteria

Oversaturate the histamine-detoxifying enzymes, enhancing the toxicity of histamine

Histamine Formed by the decarboxylation

of histidine biocatalysed by various microorganisms

Stimulates smooth muscle contraction and relaxation, including heart motions

Stimulates sensory and motory neurons

Controls acid gastric secretion

N

NH

NH2

Catalyzed reaction: R-CH2-NH2 + H2O + O2 R-CHO + NH3 + H2O2

Copper amine oxidase (AO)

Biological sources: bacteria, fungi, plants, animals

Biological functions: involved in cell growth, proliferation and differentiation

Cofactors:

Topa quinone (TPQ) Copper

Redox active polymer (PVI13-dmeOs)

Os

N

N N

NCl

N

NN

N

a

b

2+/ 3+

O

OH

O

O

NH

Cu(II)&

i

Kipp & Zonen

Sample

Potentiostat

Recorder

Flow cell

Peristaltic pump

Phosphate buffer Injection

valve

Waste

E

Flow-injection system used

AOox

AOred

Eappl.= +200 mVvs. Ag/AgCl

Electrode

2e-

NH

NCH2

CH2

NH2

NH

NCH2

CHO

OH2

NH3

Histamine

Imidazoleacetaldehyde

Working mechanism for monoenzymatic electrodes

Working mechanism for bienzymatic electrodes

NH

NCH2

CH2

NH2

NH

NCH2

OHC

OH2

NH3

Histamine

Imidazoleacetaldehyde

AOox

AOred

(TOPA -native)

(TOPA -inactive)

H2O2

O2

OH2

HRPred

HRPox

(Fe3+- native)

(Fe4+ = O, P+ inactive)

2e- Eappl.= -50 mVvs. Ag/AgCl

Electrode

2 Os2+

2 Os3+

2e-

Electrode type C

Electrode type DNo Os2+/3+

Biosensors characteristics

ELECTRODE

TYPE

ANALYTE Kmapp

(µM)Imax(µA)

S(mA/Mcm2)

DL(µM)

DR(µM)

TYPE A HISTAMINE 375±34 0.164±0.06 5.99±0.09 2.7 10-100

TYPE B HISTAMINE 730±33 0.360±0.08 6.76±0.05 2.2 10-200

TYPE C HISTAMINE

PUTRESCINE

H2O2

332±17227±16112±8

1.34±0.023.01±0.072.70±0.06

55.29±0.73181.64±1.01330.14±1.02

0.160.06

1-1001-1001-100

TYPE D HISTAMINE

PUTRESCINE

H2O2

901±85512±40977±92

4.85±0.417.26±0.5322.8±1.68

73.74±1.73194.11±1.37319.59±1.63

0.330.17

1-1501-4001-250

Native enzyme Km - putrescine 0.2 mM, histamine 0.35 mM

Monitoring real samples - turbot fish

0

5

10

15

20

25

30

0 2 4 6 8 10 12

fish kept at -20°Cfish kept at 25°C

•Amine content from fish kept in different conditions was extracted with 0.1M potassium phosphate buffer, pH 7.2, and analyzed by direct injection in the FIA system using the bienzymatic mediated electrode

g h

ista

min

e/k

g fi

sh

Days

0

50

100

150

200

250

300

350AO biosensor

AO-HRP biosensor

Rel

ativ

e re

spon

se (

%)

Amine substrate

Hsm

Csm Trm

Spd

ED

A

Agm Put

Cad

CD

AB

TD

AB

Comparison of selectivity of the developed systems

AOox

AOred

Eappl.= +200 mVvs. Ag/AgCl

Electrode

2e-

H2O2

O2

NH

NCH2

CH2

NH2

NH

NCH2

CHO

OH2

NH3

Histamine

Imidazoleacetaldehyde

NH

NCH2

COOH

Imidazoleacetic acid

+

+

Further oxidation of the histamine reaction product

Putrescine and cadaverine form cyclic products - cannot be further oxidized !!!

NH3

+

NH2

NH2

NH3

+

+ O2 + H2O

+ O2 + H2O

AO

AO

N

N

+ H2O2 + NH4+

+ H2O2 + NH4+

Putrescine

1-Pyrroline

Cadaverine

1-Piperideine

- H2O

NH2

O

- H2O

O

NH2

1. Combination of the monoenyzmatic (AO) and bienzymatic (AO-

HRP) electrode can be used for selective detection of histamine and

diamines (putrescine and cadaverine).

2. The biosensors were tested for detection of fish product poisoning

by putrefactive amines.

3. The monoenzymatic electrode (AO) is the first example of DET

with copper amine oxidase, which can proceed anaerobically.

4. With histamine as an analyte, both DET and further oxidation of

the product aldehyde contribute to the biosensor response.

Conclusions