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MICROCYSTIN DETECTIONMICROCYSTIN DETECTION
Introduction
A real problem…
… 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
TheThe “ “wonderfulwonderful” cyanobacteria blooms” cyanobacteria blooms
Grandview Garden Park, Beijing
Baltic sea
Introduction
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
Microcystin: heptapeptideMicrocystin: heptapeptideIntroduction
Microcystis aeruginosa
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
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
Enzyme sensor strategyEnzyme sensor strategy
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
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
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
Ascorbic acid (comm. or ALP)Ascorbic acid (comm. or ALP)
No fouling (CV/CA)
Electrochemical Results
Ascorbic acid (PP)Ascorbic acid (PP)
NOTHING...
Electrochemical Results
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
4-Methoxyphenol (comm. or ALP)4-Methoxyphenol (comm. or ALP)
Fouling (CV/CA)
Electrochemical Results
4-Methoxyphenol (PP)4-Methoxyphenol (PP)
NOTHING...
Electrochemical Results
Phenyl phosphatePhenyl phosphate
Ascorbic acid 2-phosphate Ascorbic acidPhenol
Protein Phosphatase
Quinonee-
Phenyl Phosphate +550mV
Electrochemical Results
Phenol (comm. or ALP)Phenol (comm. or ALP)
Fouling (CV/CA)
Electrochemical Results
Phenyl phosphate (PP)Phenyl phosphate (PP)
NOTHING...
Electrochemical Results
Naphthyl phosphateNaphthyl phosphate
Ascorbic acid 2-phosphate Ascorbic acid Naphthol (red)
Protein Phosphatase
Naphthol (ox)e-
Naphthyl Phosphate +200mV
Electrochemical Results
Naphthol (comm. or ALP)Naphthol (comm. or ALP)
Fouling (CV/CA)
Electrochemical Results
Naphthol (PP2A-Upstate)Naphthol (PP2A-Upstate)
PP2A recognises -NP by CV, but there is
fouling
PP2A = 9.1mU[-NP] = 3mM
Electrochemical Results
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
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
p-p-AminophenolAminophenol (ALP) (ALP)
Instability
Electrochemical Results
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
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
CatecholCatechol (comm. or ALP) (comm. or ALP)
Fouling (CV/CA)
Electrochemical Results
Catechol (PP2A-Upstate)Catechol (PP2A-Upstate)
PP2A recognises CMP by CV, but there is fouling
PP2A = 13.6mU[CMP] = 0.5mM
Electrochemical Results
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
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...
Electrochemical substratesEnzyme sensor
P OH
OH
OCatechyl monophosphate
4-Methylumbelliferylphosphate
α-Naphthylphosphate
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
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
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
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.
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
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
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
ImmunosensorImmunosensor strategystrategy
The “immuno” strategy
Screen-printed electrode
MC-enzymeconjugate
Enzyme product
Immunosensor
PAb/MAb
Enzyme substrate
MC
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
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)
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
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)
MPMSImmunosensor
MPMS + HRP + H2O2
MPMS Chronoamperometry
2 min substrate incubationEreading = - 0.2 V for 20 sec
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
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)
TCNQImmunosensor
Chronoamperometry
2 min substrate incubationEreading = - 0.2 V for 20 sec
TCNQ + HRP + H2O2
TCNQ
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
Aptasensor strategyAptasensor strategy
Aptasensor
Screen-printed electrode
Biotinylatedaptameragainst MC
Enzyme substrate
Enzyme product
MC-enzymeconjugate
Streptavidinor avidin
Aptasensor schemeAptasensor scheme
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