microcystin detection
DESCRIPTION
MICROCYSTIN DETECTION. Introduction. A real problem …. Introduction. … with social implications …. “Un chien mort dans les Gorges du Tarn” Lozère online , 29 juin 2005 “L’eau des gorges du Tarn empoisonne les chiens” L’indépendant, 9 août 2005 “Morts mystérieuses dans le Tarn” - PowerPoint PPT PresentationTRANSCRIPT
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MICROCYSTIN DETECTIONMICROCYSTIN DETECTION
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Introduction
A real problem…
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… with social implications… “Un chien mort dans les Gorges du Tarn”
Lozère online, 29 juin 2005
“L’eau des gorges du Tarn empoisonne les chiens”L’indépendant, 9 août 2005
“Morts mystérieuses dans le Tarn”Nuovo , 17 août 2005
“Fin de l'énigme sur la mort des chiens dans les gorges du Tarn”
Le Nouvel Observateur, 5 août 2005
Introduction
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TheThe “ “wonderfulwonderful” cyanobacteria blooms” cyanobacteria blooms
Grandview Garden Park, Beijing
Baltic sea
Introduction
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What do microcystins do?What do microcystins do?
In animals: skin sensitisation, paralysis, In animals: skin sensitisation, paralysis, convulsions, liver damage, disorientation, convulsions, liver damage, disorientation, constipation, scours, abortion and death.constipation, scours, abortion and death.
In humans: skin and eye irritation, dermatitis, In humans: skin and eye irritation, dermatitis, gastroenteritis, diarrhoea and vomiting, nausea, gastroenteritis, diarrhoea and vomiting, nausea, headaches and even death.headaches and even death.
Introduction
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Microcystin: heptapeptideMicrocystin: heptapeptideIntroduction
Microcystis aeruginosa
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Detection methodsDetection methods
METHODMETHOD TECHNIQUETECHNIQUE ADVANTAGESADVANTAGES DRAWBACKSDRAWBACKS
BiologicalBiological Mice and cellsMice and cells Easy, low-costEasy, low-cost Ethical, non specific Ethical, non specific non-sensitivenon-sensitive
ChemicalChemical HPLC-UV, HPLC-UV, LCMSLCMS
Sensitive, Sensitive, specificspecific
Expensive, long, skilled Expensive, long, skilled personnelpersonnel
ImmunologicImmunologicalal
ELISA with Mab ELISA with Mab and Paband Pab
Fast, easy, Fast, easy, sensitive, sensitive, availableavailable
Cross-reactivityCross-reactivity
EnzymaticEnzymatic PP inhibitionPP inhibitionFast, easy, Fast, easy, sensitive, sensitive, robustrobust
Non-specific, other Non-specific, other inhibitorsinhibitors
Introduction
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Our goalOur goal
MicrocystinsMicrocystins detection in drinking waterdetection in drinking water WHO: 1WHO: 1g/L microcystin-LRg/L microcystin-LR
Amperometric biosensorAmperometric biosensor
Cost-effective (SP), sensitive and reliable deviceCost-effective (SP), sensitive and reliable device
3 approaches: enzyme sensor, 3 approaches: enzyme sensor, immunosensor and aptasensorimmunosensor and aptasensor
Objective
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Enzyme sensor strategyEnzyme sensor strategy
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Locks and keysLocks and keysProtein Phosphatase : Protein Phosphatase :
production and purificationproduction and purification
Electrochemically active substrate after Electrochemically active substrate after dephosphorylationdephosphorylation
Protein Phosphatase immobilisation:Protein Phosphatase immobilisation: sol-gel, glutaraldehyde, PVA-SbQsol-gel, glutaraldehyde, PVA-SbQ
Biosensor developmentBiosensor development
Biosensor validationBiosensor validation
Objective
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Protein PhosphatasesProtein PhosphatasesEnzymatic Activity Results
Ascorbic acid 2-phosphatep-Nitrophenol + colour at = 405nm
Protein Phosphatase
p-Nitrophenyl Phosphate
PP2A-Upstate: PP2A-Upstate: 1900 mU / mL1900 mU / mL
PP1-Biolabs:PP1-Biolabs: 1574 mU / mL1574 mU / mL
PP2A-GTP: PP2A-GTP: 1080 mU / mL1080 mU / mL
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Ascorbic acid 2-phosphateAscorbic acid 2-phosphate
P OH
OH
O
Ascorbic acid 2-phosphate Ascorbic acidAscorbic Acid (red)
Protein Phosphatase
Ascorbic Acid (ox)e-
Ascorbic Acid 2-Phosphate +400mV
Electrochemical Results
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Ascorbic acid (comm. or ALP)Ascorbic acid (comm. or ALP)
No fouling (CV/CA)
Electrochemical Results
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Ascorbic acid (PP)Ascorbic acid (PP)
NOTHING...
Electrochemical Results
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4-Methoxyphenyl phosphate4-Methoxyphenyl phosphate
Ascorbic acid 2-phosphate Ascorbic acid4-Methoxyphenol (red)
Protein Phosphatase
4-Methoxyphenol (ox)e-
4-Methoxyphenyl Phosphate
+350mV
NMR: Non-pure P OH
OH
O
Electrochemical Results
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4-Methoxyphenol (comm. or ALP)4-Methoxyphenol (comm. or ALP)
Fouling (CV/CA)
Electrochemical Results
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4-Methoxyphenol (PP)4-Methoxyphenol (PP)
NOTHING...
Electrochemical Results
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Phenyl phosphatePhenyl phosphate
Ascorbic acid 2-phosphate Ascorbic acidPhenol
Protein Phosphatase
Quinonee-
Phenyl Phosphate +550mV
Electrochemical Results
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Phenol (comm. or ALP)Phenol (comm. or ALP)
Fouling (CV/CA)
Electrochemical Results
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Phenyl phosphate (PP)Phenyl phosphate (PP)
NOTHING...
Electrochemical Results
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Naphthyl phosphateNaphthyl phosphate
Ascorbic acid 2-phosphate Ascorbic acid Naphthol (red)
Protein Phosphatase
Naphthol (ox)e-
Naphthyl Phosphate +200mV
Electrochemical Results
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Naphthol (comm. or ALP)Naphthol (comm. or ALP)
Fouling (CV/CA)
Electrochemical Results
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Naphthol (PP2A-Upstate)Naphthol (PP2A-Upstate)
PP2A recognises -NP by CV, but there is
fouling
PP2A = 9.1mU[-NP] = 3mM
Electrochemical Results
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Naphthol (PP1-Biolabs)Naphthol (PP1-Biolabs)
PP1 recognises-NP by CA, but there is fouling
PP1 = 7.5 mU[-NP] = 10mM
E = +370mVt = 9min
116nA(blk: 5nA)
Electrochemical Results
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p-p-AminophenylAminophenyl phosphate phosphate
Ascorbic acid 2-phosphate Ascorbic acidp-Aminophenol (red)
Protein Phosphatase
p-Aminophenol (ox)e-
p-Aminophenyl Phosphate +150mV
Electrochemical Results
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p-p-AminophenolAminophenol (ALP) (ALP)
Instability
Electrochemical Results
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p-Aminophenol (PP1-Biolabs)p-Aminophenol (PP1-Biolabs)
PP1 recognisesp-APP by CA,
but p-AP is unstable
PP1 = 10mU[p-APP] = 0.1mM
E = +150mVt = 15min
67nA(blk: 5nA)
Electrochemical Results
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Catechyl monophosphateCatechyl monophosphate
Ascorbic acid 2-phosphate Ascorbic acidCatechol (red)
Protein Phosphatase
Catechol (ox)e-
Catechyl Monophosphate +40mV
NMR: Catechyl monophosphate P OH
OH
O
Electrochemical Results
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CatecholCatechol (comm. or ALP) (comm. or ALP)
Fouling (CV/CA)
Electrochemical Results
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Catechol (PP2A-Upstate)Catechol (PP2A-Upstate)
PP2A recognises CMP by CV, but there is fouling
PP2A = 13.6mU[CMP] = 0.5mM
Electrochemical Results
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Catechol (PP1-Biolabs)Catechol (PP1-Biolabs)
PP1 recognises CMP by CA!!!,
but there is fouling
PP1 = 7.5mU[CMP] = 5mME = +450mV
t = 9min
Electrochemical Results
1383nA(blk: 395nA)
CMP + PP
blank
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Natural susbstrates: peptidesNatural susbstrates: peptides
Ascorbic acid 2-phosphate Ascorbic acidRRACVA Peptide (red)
Protein Phosphatase
RRACVA Peptide (ox)e-
RRApCVA Peptide +500mV
Electrochemical Results
Ascorbic acid 2-phosphate Ascorbic acidRRAYVA Peptide (red)
Protein Phosphatase
RRAYVA Peptide (ox)e-
RRApYVA Peptide +550mV
DIFFICULT SYNTHESIS...
NOTHING...
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Electrochemical substratesEnzyme sensor
P OH
OH
OCatechyl monophosphate
4-Methylumbelliferylphosphate
α-Naphthylphosphate
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CV and amperometryEnzyme sensor
α-Naphthyl phosphate + PP: + 300 mV → 116 nA (blk: 23%)
Catechyl phosphate + PP: + 450 mV → 637 nA (blk: 5%)
4-Methylumbelliferyl phosphate + PP: + 700 mV → 429 nA (blk: 52%)
MUP + PP
blank
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H3 CN+
O
OOH
OH
OH
OH
N+CH3
OO
OHOH
OHOH
PVA-SbQprepolymer
h ν
E
E
E
Y -N+CH3
OO
OHOH
OHOH
(Enzyme)
+ E E
PVA-SbQ entrapment method
Immobilisation Results
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MC-LR detection (e-)Enzyme sensor
PP:PVA
3 h neon light1 day drying
4°C
Inhibition
30 min MCRT
Electrochemical detection
5mM CP+ 450 mV
C W R
IC50 = 8.30 μg/L
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PP1α
• genetically engineered enzyme
• histidine tags
•selective towards MCs
The affinity of histidine residues for Ni precharged magnetic beads allows selective immobilisation of histidine fusion protein.
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30 mg of PP1α
25 μL of mag-Ni-PP1α in 300 μL assay buffer
30 μL/SPE+160 μL of MC-LR
30’ incubation
+10 μL α-NPP
SPE
Chronoamperometry
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MC-LR, ppb
1 10 100 1000
I, %
40
50
60
70
80
90
100
110
IC50=12 ppb
IC50=77 ppb
(M. Campàs et al., 2005)
PP1PP1αα BIOSENSOR BIOSENSOR
SPE
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Random sequence
Constantregion
Constantregion
5’ 3’
ssDNA oligonucleotides
Combinatorial LibraryTarget
OTAMagnetic
Beads
Binding
Washing
Elution
AmplificationPCR
Purification of ssDNA
dsDNA
Enriched ssDNA pool
1. SELEXRound
2-n SELEXRound
Last SELEXRound
Cloning andSequencing
SELEXProcess
MB
MB
MB
MB
MB
Removal of unboundoligonucleotides
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ImmunosensorImmunosensor strategystrategy
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The “immuno” strategy
Screen-printed electrode
MC-enzymeconjugate
Enzyme product
Immunosensor
PAb/MAb
Enzyme substrate
MC
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CheckerboardsImmunosensor
[MAb] = 1 µg/L [MC-LR-HRP]MAb = 195,0 µg/L
[PAb] = 1:2,750 [MC-LR-HRP]PAb = 277,5 µg/L
21,9 µg/L
23,5 µg/L
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Competition optimisationImmunosensor
MC-LR-HRP incubation time: compromise betweenthe colorimetric response (from HRP) and the [MC]
2 h MC incubation + 30 min competition with MC-LR-HRP (90 µL of MC + 10 µL of MC-LR-HRP)
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MC-LR detection (colour)
ELISA WELLS
IC50 (MAb) = 0.14 μg/L
IC50 (PAb) = 1.60 μg/L
SPEs
IC50 (MAb) = 0.28 μg/L
IC50 (PAb) = 1.81 μg/L
Immunosensor
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Looking for a mediatorImmunosensor
Ferrocene carboxylic acid
o-Phenylene diamine (PDA)
Catechol
2,6-Dichlorophenol-indophenol (DPIP)
Os(2,2‘-bipyridyl)2Cl(4-(aminomethyl)pyridine)
7,7,8,8-Tetracyanoquinodimethane (TCNQ)
1-Methoxy-5-methyl-phenazinium methyl sulfate (MMPMS)
5-Methyl-phenazinium methyl sulfate (MPMS)
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MPMSImmunosensor
MPMS + HRP + H2O2
MPMS Chronoamperometry
2 min substrate incubationEreading = - 0.2 V for 20 sec
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MC-LR detection (e-)Immunosensor
MPMS in solution
Total system for MAb: 5441 ± 542 nA (10.0 %)
Total system for PAb: 5698 ± 675 nA (11.9 %)
No Ab: 4595 ± 362 nA (7.9 %)No MC-LR-HRP: 4400 ± 342 nA (7.8 %)No H2O2: 2901 ± 115 nA (4.0 %)No MPMS: 1026 ± 183 nA (17.8 %)
IC50 (MAb) = 0.02 μg/L
IC50 (PAb) = 1.73 μg/L
19 % for MAb
15 % for PAb
of MC-LR-HRP
non-specific
adsorption
1041
1298
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Looking for an immobilised mediator
Immunosensor
Ferrocene-COOH
Catechol
1-Methoxy-5-methyl-phenazinium methyl sulfate (MMPMS)
Prussian Blue (PB)
Meldola Blue Reinecke salt (MBRS)
Os “wire”
Cobalt phthalocyanine
7,7,8,8-Tetracyanoquinodimethane (TCNQ)
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TCNQImmunosensor
Chronoamperometry
2 min substrate incubationEreading = - 0.2 V for 20 sec
TCNQ + HRP + H2O2
TCNQ
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MC-LR detection (e-)Immunosensor
Immobilised TCNQ
Total system for MAb: 2404 ± 172 nA (7.2 %)
Total system for PAb: 2770 ± 399 nA (14.4 %)
No Ab: 2021 ± 152 nA (7.5 %)No MC-LR-HRP: 1912 ± 196 nA (10.3 %)No H2O2: 448 ± 43 nA (9.6 %)No TCNQ: 802 ± 61 nA (7.6 %)
22 % for MAb
13 % for PAb
of MC-LR-HRP
non-specific
adsorption
IC50 (MAb) = 0.46 μg/L
IC50 (PAb) = 1.66 μg/L
492
858
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Aptasensor strategyAptasensor strategy
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Aptasensor
Screen-printed electrode
Biotinylatedaptameragainst MC
Enzyme substrate
Enzyme product
MC-enzymeconjugate
Streptavidinor avidin
Aptasensor schemeAptasensor scheme
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Random sequence
Constantregion
Constantregion
5’ 3’
ssDNA oligonucleotides
Combinatorial LibraryTarget
OTAMagnetic
Beads
Binding
Washing
Elution
AmplificationPCR
Purification of ssDNA
dsDNA
Enriched ssDNA pool
1. SELEXRound
2-n SELEXRound
Last SELEXRound
Cloning andSequencing
SELEXProcess
MB
MB
MB
MB
MB
Removal of unboundoligonucleotides