MICROCYSTIN 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

The “wonderful” cyanobacteria blooms Grandview Garden Park, Beijing Baltic sea Introduction

What do microcystins do?  In animals: skin sensitisation, paralysis, convulsions, liver damage, disorientation, constipation, scours, abortion and death.  In humans: skin and eye irritation, dermatitis, gastroenteritis, diarrhoea and vomiting, nausea, headaches and even death. Introduction

Microcystin: heptapeptide Introduction Microcystis aeruginosa

Detection methods METHODTECHNIQUEADVANTAGESDRAWBACKS Biological Mice and cells Easy, low-cost Ethical, non specific non-sensitive Chemical HPLC-UV, LCMS Sensitive, specific Expensive, long, skilled personnel Immunologic al ELISA with Mab and Pab Fast, easy, sensitive, available Cross-reactivity Enzymatic PP inhibition Fast, easy, sensitive, robust Non-specific, other inhibitors Introduction

Our goal  Microcystins detection in drinking water WHO: 1  g/L microcystin-LR  Amperometric biosensor  Cost-effective (SP), sensitive and reliable device  3 approaches: enzyme sensor, immunosensor and aptasensor Objective

Enzyme sensor strategy

Locks and keys  Protein Phosphatase : production and purification  Electrochemically active substrate after dephosphorylation  Protein Phosphatase immobilisation: sol-gel, glutaraldehyde, PVA-SbQ  Biosensor development  Biosensor validation Objective

Protein Phosphatases Enzymatic Activity Results Ascorbic acid 2-phosphate p-Nitrophenol + colour at = 405nm Protein Phosphatase p-Nitrophenyl Phosphate  PP2A-Upstate: 1900 mU / mL  PP1-Biolabs:1574 mU / mL  PP2A-GTP: 1080 mU / mL

Ascorbic acid 2-phosphate P OH O Ascorbic acid 2-phosphate Ascorbic acid Ascorbic Acid (red) Protein Phosphatase Ascorbic Acid (ox) e-e- Ascorbic Acid 2-Phosphate +400mV Electrochemical Results

Ascorbic acid (comm. or ALP) No fouling (CV/CA) Electrochemical Results

Ascorbic acid (PP) NOTHING... Electrochemical Results

4-Methoxyphenyl phosphate Ascorbic acid 2-phosphate Ascorbic acid 4-Methoxyphenol (red) Protein Phosphatase 4-Methoxyphenol (ox) e-e- 4-Methoxyphenyl Phosphate +350mV NMR: Non-pure P OH O Electrochemical Results

4-Methoxyphenol (comm. or ALP) Fouling (CV/CA) Electrochemical Results

4-Methoxyphenol (PP) NOTHING... Electrochemical Results

Phenyl phosphate Ascorbic acid 2-phosphate Ascorbic acid Phenol Protein Phosphatase Quinone e-e- Phenyl Phosphate +550mV Electrochemical Results

Phenol (comm. or ALP) Fouling (CV/CA) Electrochemical Results

Phenyl phosphate (PP) NOTHING... Electrochemical Results

 Naphthyl phosphate Ascorbic acid 2-phosphate Ascorbic acid  Naphthol (red) Protein Phosphatase  Naphthol (ox) e-e-  Naphthyl Phosphate +200mV Electrochemical Results

 Naphthol (comm. or ALP) Fouling (CV/CA) Electrochemical Results

 Naphthol (PP2A-Upstate) PP2A recognises  - NP by CV, but there is fouling PP2A = 9.1mU [  -NP] = 3mM Electrochemical Results

 Naphthol (PP1-Biolabs) PP1 recognises  -NP by CA, but there is fouling PP1 = 7.5 mU [  -NP] = 10mM E = +370mV t = 9min 116nA (blk: 5nA) Electrochemical Results

p- Aminophenyl phosphate Ascorbic acid 2-phosphate Ascorbic acid p-Aminophenol (red) Protein Phosphatase p-Aminophenol (ox) e-e- p-Aminophenyl Phosphate +150mV Electrochemical Results

p- Aminophenol (ALP) Instability Electrochemical Results

p-Aminophenol (PP1-Biolabs) PP1 recognises p-APP by CA, but p-AP is unstable PP1 = 10mU [p-APP] = 0.1mM E = +150mV t = 15min 67nA (blk: 5nA) Electrochemical Results

Catechyl monophosphate Ascorbic acid 2-phosphate Ascorbic acid Catechol (red) Protein Phosphatase Catechol (ox) e-e- Catechyl Monophosphate +40mV NMR: Catechyl monophosphate P OH O Electrochemical Results

Catechol (comm. or ALP) Fouling (CV/CA) Electrochemical Results

Catechol (PP2A-Upstate) PP2A recognises CMP by CV, but there is fouling PP2A = 13.6mU [CMP] = 0.5mM Electrochemical Results

Catechol (PP1-Biolabs) PP1 recognises CMP by CA!!!, but there is fouling PP1 = 7.5mU [CMP] = 5mM E = +450mV t = 9min Electrochemical Results 1383nA (blk: 395nA) CMP + PP blank

Natural susbstrates: peptides Ascorbic acid 2-phosphate Ascorbic acid RRACVA Peptide (red) Protein Phosphatase RRACVA Peptide (ox) e-e- RRApCVA Peptide +500mV Electrochemical Results Ascorbic acid 2-phosphate Ascorbic acid RRAYVA Peptide (red) Protein Phosphatase RRAYVA Peptide (ox) e-e- RRApYVA Peptide +550mV DIFFICULT SYNTHESIS... NOTHING...

Electrochemical substrates Enzyme sensor P OH O Catechyl monophosphate 4-Methylumbelliferyl phosphate α-Naphthyl phosphate

CV and amperometry Enzyme sensor α-Naphthyl phosphate + PP: mV → 116 nA (blk: 23%) Catechyl phosphate + PP: mV → 637 nA (blk: 5%) 4-Methylumbelliferyl phosphate + PP: mV → 429 nA (blk: 52%) MUP + PP blank

PVA-SbQ entrapment method Immobilisation Results

MC-LR detection (e - ) Enzyme sensor PP:PVA 3 h neon light 1 day drying 4°C Inhibition 30 min MC RT Electrochemical detection 5mM CP mV C W RC W R IC 50 = 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 I, % IC 50 =12 ppb IC 50 =77 ppb ( M. Campàs et al., 2005) PP1α BIOSENSOR SPE

Immunosensor strategy

The “ immuno ” strategy Screen-printed electrode MC-enzyme conjugate Enzyme product Immunosensor PAb/MAb Enzyme substrate MC

Checkerboards Immunosensor   [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 optimisation Immunosensor   MC-LR-HRP incubation time: compromise between the 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 IC 50 (MAb) = 0.14 μg/L IC 50 (PAb) = 1.60 μg/L SPEs IC 50 (MAb) = 0.28 μg/L IC 50 (PAb) = 1.81 μg/L Immunosensor

Looking for a mediator Immunosensor   Ferrocene carboxylic acid   o-Phenylene diamine (PDA)   Catechol   2,6-Dichlorophenol-indophenol (DPIP)   Os(2,2‘-bipyridyl) 2 Cl(4-(aminomethyl)pyridine)   7,7,8,8-Tetracyanoquinodimethane (TCNQ)   1-Methoxy-5-methyl-phenazinium methyl sulfate (MMPMS)   5-Methyl-phenazinium methyl sulfate (MPMS)

MPMS Immunosensor MPMS + HRP + H 2 O 2 MPMS Chronoamperometry 2 min substrate incubation E reading = 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 H 2 O 2 :2901 ± 115 nA (4.0 %) No MPMS:1026 ± 183 nA(17.8 %) IC 50 (MAb) = 0.02 μg/L IC 50 (PAb) = 1.73 μg/L 19 % for MAb 15 % for PAb of MC-LR-HRP non-specific adsorption

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)

TCNQ Immunosensor Chronoamperometry 2 min substrate incubation E reading = V for 20 sec TCNQ + HRP + H 2 O 2 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 H 2 O 2 : 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 IC 50 (MAb) = 0.46 μg/L IC 50 (PAb) = 1.66 μg/L

Aptasensor strategy

Aptasensor Screen-printed electrode Biotinylated aptamer against MC Enzyme substrate Enzyme product MC-enzyme conjugate Streptavidin or avidin Aptasensor scheme