Transcript Quinoproteins, a novel grop of enzymes containing

Biosensors
A focus on peroxidase-modified electrodes and their practical
applications
by
Ivo Frébort
Biosensor
- an analytical device that exploits a biocatalytic reaction
Consists of:
biocatalyst (enzyme, cells, tissue)
transducer (converts the biological or biochemical signal
into a quantifiable electrical or optical signal)
First biosensor - Clark (1962):
glucose sensor with glucose oxidase and oxygen electrode
Glucose + O2
Gluconic acid + H2O2
Oxygen electrode (1956)
working electrode: Pt cathode (-0.6 V)
reference electrode: Ag/AgCl
electrodes separated from measured
solution with a gas permeable mebrane
Leland C. Clark, Jr. with the first enzyme electrode
Construction of the biosensors
Sensing electrode: platinum, gold, various forms of carbon
Immobilization techniques: general method doesn’t exist
- enzyme physical entrapment
- covalent crosslinking
O CH
BSA
o-ring
E E E
E
E
dialyzing
membrane
E
BSA
Glutaraldehyde
E
BSA
electrode
E
CH O
+ H2N
Schiff base
BSA
E
(CH2)3 CH O
CH N
BSA
Reduction with NaBH4
CH2 NH
Covalent attachment to a support membrane or the electrode
R
R
N
O
C
COOH + C
N
N
B
+
O C
NH
R'
Carbodiimide reaction
NH2
R'
O
C
NH
R
NH
B
C O
DCC
N C N
R'
NH
N C N CH2CH2 N
H3C
CH3CH2
O CH
(CH2)3 CH O
N CH
NH2
N C N
CMC
O
tosyl-
(CH2)3 N(CH3)2 EDC
(CH2 )3 CH O
B
NH2
NaBH4
N CH
(CH2)3 CH N
Glutaraldehyde reaction
B
NH CH
(CH2)3 CH NH
B
Adsorption - glass, carbon, Au, Pt
- often activation needed
Adsorption of thiols to a gold electrode
+
S
S
Au
S
(CH2)2 NH2
S
(CH2)2 NH2
(CH2)2 NH2
(CH2)2 NH2
Silanization of an oxidized metal electrode
Au
O
R Si(OC2H5)3
--OH
O Si R
O
--OH
O Si R
O
S
S
S
S
S
S
Organized layer
X
X
X
X
X
X
S
S
S
S
S
S
X
X
Dilution with an inert thiol
(C 2H5)3Si
(CH3)3Si
(CH2)3 NH2
(CH2)3 O CH2
CH3
C 2H5 O Si (CH2)3 NH2
CH3
APTES
CH CH2
O
GOPS
APDMES
Screen printing
Mobile wiper
Matrix carrier
Paste
Screen grid
Screen print
An enzyme electrode
P1
P1
S1
-
-
Sample
E
Electrode
S2
-
P2
I
Protective
membrane
P2
-
P2*
Enzyme layer
Permselective
membrane
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 slow
Enzyme activity high - thin membrane OK, rapid response
3. Detection: Steady-state or flow injection analysis
Biosensor parameters
1. Sensitivity
2. Linear response
3. Detection limit
4. Background noise
5. Baseline drift
6. Selectivity
7. Response
8. Operating stability
9. Shelf life
dS/dt
Analyte
Signal
DS
Background noise
Time
S
Analyte
Detection
limit
N
DS/Dc Linear response
S=3N
Assay of the detection limit
c
Type of measurement
Analyte additions
S
Time
Direct contact with the sample
Solution placed in a chamber
A
Flow cell
S
Time
IN
OUT
B
First generation biosensors - response to the substrates in solution
Glucose + O2
Gluconic acid + H2O2
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
Examples of hydrogen peroxide measurning biosensor
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
Glucose oxidase
Starch + H2O  β-D-Glucose
β-D-Glucose + O2  Gluconic acid + H2O2
Sucrose
Invertase
Mutarotase
Glucose oxidase
Sucrose + H2O  α-D-Glucose + β-D-Fructose
α-D-Glucose  β-D-Glucose
β-D-Glucose + O2  Gluconic acid + H2O2
Types of transducers used in biosensors
Type
Amperometric
Detectable species
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 miniaturized
blood 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 bioluminiscence
human chorionic gonadotropin - pregnancy
Examples of biosensors
Analyte
Biocatalyst
Transducer
Alcohol
Alcohol oxidase
Arginine
Streptococcus faecium
Cholesterol Nocardia erythropolis
D-Glucose Glucose oxidase
Glutamate
Glutamate decarboxylase
NAD+
NADase + Escherichia coli
Nitrate
Azotobacter vinelandii
Penicillin
Penicillinase
Urea
Urease
O2
NH3
O2
O2
CO2
NH3
NH3
H+
NH4+
Immobilization
Shelf life
Glutaraldehyde
2 weeks
Entrapment
3 weeks
Entrapment
4 weeks
Covalent
3 weeks
Glutaraldehyde
1 week
Membrane
1 week
Entrapment
2 weeks
Polyacrylamide
2 weeks
Polyacrylamide
3 weeks
Response
1-2 min
20 min
35-70 s
1 min
10 min
5-10 min
7-8 min
15-30 s
20-40 s
Personal glucose meter for diabetics
(Medisense Britain, Ltd.)
Automated affinity systems
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
Peroxidase-based electrodes
PEROXIDASE (EC 1.11.1.7)
Protein of 35-45 kDa, prosthetic
group - heme, Mn2+
Convenient sources:
horse radish root, soybean,
tobacco leaves, various fungi
Ruiz-Duenas, F. J., Martinez, M. J., Martinez, A. T.: Peroxidase from the ligninolytic fungus
Pleurotus eryngii. Mol Microbiol 31 pp. 223 (1999)
The catalytic cycle of peroxidase
Native peroxidase + H2O2
(Fe3+)
Compound-I + AH2
(Fe4+=O, Por+)
Compound-II + AH2
(Fe4+=O)
Compound-I + H2O
(Fe4+=O, Por+)
Compound-II + AH*
(Fe4+=O)
Native peroxidase+ AH* + H2O
(Fe3+)
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
Electrode designs
A. Surface modified electrodes
Electrode material: graphite, glassy carbon, gold, SnO2
Coupling: carbodiimide, glutaraldehyde, adsorption
B. Polymer-based electrodes
Crosslinking with Os(bpy)23+/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
Mechanism of direct biocatalytic reduction of
hydrogen peroxide at peroxidase-modified electrodes
H2O
Compound-I
(Fe 4+=O, Por+)
e-
Compound-II
(Fe 4+=O)
2H+
Electrode
Eappl< 0.6 V
e-
H2O2
H2O
Ferriperoxidase
(Fe 3+)
vs SCE
Mechanism of mediated biocatalytic reduction of
hydrogen peroxide at peroxidase-modified electrodes
H2O
Compound-I
(Fe 4+=O, P +)
Mred
Mox
Compound-II
(Fe 4+=O)
2H +
H2O2
H 2O
Electrode
Mred
Mox
Ferriperoxidase
(Fe 3+)
Mediator: ferrocene, o-phenylenediamine, hydroquinone
The mediators
OH
R
NH2
+
Fe
NH2
Ferrocene
o-Phenylenediamine
OH
Hydroquinone
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, cadaverine
Currently 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 use
Aim of the work: design and construction of the amperometric
biosensors for monitoring of biomarkers
Two biosensor designs: monoenzymatic and bienzymatic, using
both the direct and mediated electron transfer pathways
Biological 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
NH2
N
N
H




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
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 histaminedetoxifying enzymes, enhancing
the toxicity of histamine
Copper amine oxidase (AO)
Redox active polymer
(PVI13-dmeOs)



Biological sources: bacteria, fungi,
plants, animals
Biological functions: involved in cell
growth, proliferation and differentiation
Cofactors:
O
H
N
O
b
N
N
N
N
N
&
HO
a
N
Os2+/ 3+
Cu(II)
N
Cl N
O
Topa quinone (TPQ)
Copper
Catalyzed reaction: R-CH2-NH2 + H2O + O2  R-CHO + NH3 + H2O2
Flow-injection system used
Working mechanism for monoenzymatic electrodes
H
N
N
H2
C C
H2
NH2
Histamine
Electrode
AOox
NH3
H2O
H
N
AOred
N
C CHO
H2
Imidazoleacetaldehyde
2e-
Eappl.= +200 mV
vs. Ag/AgCl
Working mechanism for bienzymatic electrodes
Electrode
H
N
H2
N
C C
H2O
H2
H2N
Histamine
NH3
H
N
N
AOox
(TOPA -native)
H2O2
H2O
AOred
(TOPA -inactive)
HRPred
(Fe3+- native)
HRPox
(Fe4+ =
2e-
2 Os2+
2 Os3+
O, P+ inactive)
O2
OHC C
H2
Imidazoleacetaldehyde
Electrode type C
Electrode type D
No Os2+/3+
2e-
Eappl.= -50 mV
vs. Ag/AgCl
Biosensors characteristics
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±17
227±16
112±8
1.34±0.02
3.01±0.07
2.70±0.06
55.29±0.73
181.64±1.01
330.14±1.02
0.16
0.06
1-100
1-100
1-100
TYPE D
HISTAMINE
PUTRESCINE
H2O2
901±85
512±40
977±92
4.85±0.41
7.26±0.53
22.8±1.68
73.74±1.73
194.11±1.37
319.59±1.63
0.33
0.17
1-150
1-400
1-250
ELECTRODE
TYPE
Native enzyme Km - putrescine 0.2 mM, histamine 0.35 mM
Monitoring real samples - turbot fish
30
g histamine/kg fish
fish kept at -20°C
fish kept at 25°C
25
20
15
10
5
0
0
2
4
6
8
10
12
Days
•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
Comparison of selectivity of the developed systems
350
AO biosensor
AO-HRP biosensor
250
200
150
100
TDAB
Cad
CDAB
Amine substrate
Put
Agm
EDA
Spd
Trm
0
Csm
50
Hsm
Relative response (%)
300
Further oxidation of the histamine reaction product
H2O
+
O2
H
N
AOred
H2
C C
H2
NH2
Histamine
N
NH3
+ H2O2
H
N
Electrode
AOox
N
C CHO
H2
Imidazoleacetaldehyde
H
N
N
C COOH
H2
Imidazoleacetic acid
2e-
Eappl.= +200 mV
vs. Ag/AgCl
+
NH3
NH2
AO
+ O2 + H2O
NH2
Putrescine
+
NH2
Cadaverine
+ H2O2 + NH4+
- H2O
Putrescine and cadaverine form
cyclic products - cannot be
further oxidized !!!
NH3
O
N
1-Pyrroline
AO
+ O2 + H2O
O
+ H2O2 + NH4+
NH2
- H2O
N
1-Piperideine
Conclusions
1. Combination of the monoenyzmatic (AO) and bienzymatic (AOHRP) 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.