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MICROCYSTIN DETECTION

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”

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MICROCYSTIN DETECTION

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  1. MICROCYSTIN DETECTION

  2. Introduction A real problem…

  3. 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” Nuovo , 17 août 2005 • “Fin de l'énigme sur la mort des chiens dans les gorges du Tarn” Le Nouvel Observateur, 5 août 2005

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

  5. 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.

  6. Introduction Microcystin: heptapeptide Microcystis aeruginosa

  7. Introduction Detection methods

  8. Objective Our goal • Microcystinsdetection in drinking water WHO: 1mg/L microcystin-LR • Amperometric biosensor • Cost-effective (SP), sensitive and reliable device • 3 approaches: enzyme sensor, immunosensor and aptasensor

  9. Enzyme sensor strategy

  10. Objective 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

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

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

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

  14. Electrochemical Results Ascorbic acid (PP) NOTHING...

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

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

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

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

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

  20. Electrochemical Results Phenyl phosphate (PP) NOTHING...

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

  22. Electrochemical Results a-Naphthol (comm. or ALP) Fouling (CV/CA)

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

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

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

  26. Electrochemical Results p-Aminophenol (ALP) Instability

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

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

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

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

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

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

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

  34. MUP + PP blank Enzyme sensor CV and amperometry 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%)

  35. Immobilisation Results PVA-SbQ entrapment method

  36. CWR Electrochemical detection PP:PVA Inhibition 3 h neon light 1 day drying 4°C 30 min MC RT 5mM CP + 450 mV IC50 = 8.30 μg/L Enzyme sensor MC-LR detection (e-)

  37. 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.

  38. SPE 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 Chronoamperometry

  39. 110 100 90 80 I, % 70 60 50 40 1 10 100 1000 MC-LR, ppb SPE PP1α BIOSENSOR IC50=12 ppb IC50=77 ppb (M. Campàs et al., 2005)

  40. Immunosensorstrategy

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

  42. 21,9 µg/L 23,5 µg/L Immunosensor Checkerboards • [MAb] = 1 µg/L [MC-LR-HRP]MAb = 195,0 µg/L • [PAb] = 1:2,750 [MC-LR-HRP]PAb = 277,5 µg/L

  43. Immunosensor Competition optimisation • 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)

  44. Immunosensor 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

  45. Immunosensor Looking for a mediator • 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)

  46. MPMS MPMS + HRP + H2O2 Immunosensor MPMS Chronoamperometry 2 min substrate incubation Ereading = - 0.2 V for 20 sec

  47. IC50 (MAb) = 0.02 μg/L IC50 (PAb) = 1.73 μg/L Immunosensor MC-LR detection (e-) MPMS in solution 19 % for MAb 15 % for PAb of MC-LR-HRP non-specific adsorption 1041 1298 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 %)

  48. Immunosensor Looking for an immobilised mediator • 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)

  49. TCNQ TCNQ + HRP + H2O2 Immunosensor TCNQ Chronoamperometry 2 min substrate incubation Ereading = - 0.2 V for 20 sec

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