by ivo frébort biosensors a focus on peroxidase-modified electrodes and their practical...
TRANSCRIPT
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
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
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
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