MICROCYSTIN DETECTION. Introduction A real problem …

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