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Microcystin Detection for Water Safety: Enzyme Sensor Strategy

Explore the detection of harmful microcystins in drinking water using enzyme sensors for precise and reliable monitoring, ensuring public health safety. Learn about the detection methods, enzyme sensor development, and electrochemical results for efficient microcystin detection.

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Microcystin Detection for Water Safety: Enzyme Sensor Strategy

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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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