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Towards the development of an amperometric immunosensor for the determination of Ochratoxin A

Towards the development of an amperometric immunosensor for the determination of Ochratoxin A. Introduction. World Health Organization. Mycotoxins (from fungi) Phycotoxins (from algae) Toxins from edible plants

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Towards the development of an amperometric immunosensor for the determination of Ochratoxin A

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  1. Towards the development of an amperometric immunosensor for the determination of Ochratoxin A

  2. Introduction World Health Organization • Mycotoxins (from fungi) • Phycotoxins (from algae) • Toxins from edible plants Regulations to protect public health and economical interests of producers and traders

  3. O C O O H O O H N O H C H 3 Cl Introduction Ochratoxin A (OTA): 7-(L-b-phenylalanyl-carbonyl)-carboxyl-5-chloro-8-hydroxy-3,4-dihydro-3R-methylisocoumarin • OTA occurs when food with a high water content is not properly dried • Effects on animals: nephrotoxic, hepatotoxic, neurotoxic, immunotoxic, genotoxic, teratogenic, myelotoxic and carcinogenic (in mice and rats) properties • Effects on humans: - Group 2B (possible human carcinogen) - Main target organ: kidneys. Nephropaties

  4. Introduction OTA in foodstuffs: • Cereals (corn, barley, rice,…) and cereal products • Cocoa and cocoa products • Coffee (roasted or soluble) • Species • Dried vine fruits (currants, raisins and sultanas) • Meat and meat products • Beer • Grape juice and must • Wine 5 ppb 5-10 ppb 10 ppb 2 ppb 2 ppb

  5. Introduction Analytical methods used for OTA determination • Extraction using organic solvents • Purification through chromatographic columns • Detection HPLC with fluorescence detection GC/MS Capillary electrophoresis with fluorescence detection (induced by laser)

  6. Introduction Commercial kits for OTA based on Enzyme-Linked Immunosorbent Assays (ELISA) • Direct competitive assays R-Biopharm (LOD:5 ppb) Romer Labs (2-40 ppb LOD:2 ppb) Neogen (2-25 ppb LOD:1 ppb) • Indirect competitive assays Biosystems (1-50 ppb LOD:1 ppb)

  7. Electrochemical immunosensors more sensitive cost-effective instrumentation possible miniaturization portable device possibility of a high throughput sample SPEs Introduction Our purpose… • Immunoassays good selectivity good sensitivity wide variety of applications Wells

  8. Enzyme substrate Enzyme product IgG-enzyme conjugate anti-OTA PAb OTA anti-OTA PAb OTA-BSA Screen-printed electrode Introduction Our purpose… • Development of an immunosensor for OTA determination based on an electrochemical detection

  9. N N + O C O O H O O H N N N O O O H R1 C 10 min at room T protected from light constant stirring O H C H 3 Cl O + + CO2 N N N N H H R1 N N + O added dropwise + R2 R2 N H 2 h constant stirring R1 N 2 H OTA-BSA BSA OTA-BSA conjugate Synthesis of OTA-BSA

  10. anti-OTA PAb Enzyme substrate Enzyme product IgG-enzyme conjugate anti-OTA PAb OTA OTA-BSA Screen-printed electrode Immunosensor Immunosensor Strategy 1

  11. Immunosensor Immunosensor Checkerboard on wells • [OTA-BSA] = 0.2 µg/mL • [anti-OTA] = 1:100 • [IgG-HRP] = 1:6000 • 1 h competition (10 µL anti-OTA + 90 µL OTA) • 1 h incubation (IgG-enzyme conjugate)

  12. 1.2 1.0 0.8 IgG-HRP 0.6 IgG-AP 0.4 0.2 Abs(relative) (620 or 405 nm) 0.0 -5 -4 -3 -2 -1 0 [anti-OTA]= 2mg/ml log(dilution of IgG-enzyme conjugate) Immunosensor IgG-enzyme conjugate Saturation dilutions: - 1:1500 IgG-HRP - 1:250 IgG-AP Approx. 80%: -1:6000 dilution of IgG-HRP -1:1000 dilution of IgG-AP 0.5, 1, 4, 8, 12 and 16 mg/mL p-NPP in 10% DEA buffer, pH 9.8

  13. 100 80 IgG-AP 60 % of absorbance IgG-HRP 40 20 0 -3 -2 -1 0 1 2 log([OTA] (mg/ml)) Immunosensor Colourimetric OTA detection (ELISA wells) • IC50 (IgG/HRP) = 341 µg/L • IC30 (IgG/HRP) = 58 µg/L • IC10 (IgG/HRP) = 10 µg/L • IC50 (IgG/AP) = 1366 µg/L • IC30 (IgG/AP) = 390 µg/L • IC10 (IgG/AP) = 111 µg/L n = 4

  14. Immunosensor Immunosensor Checkerboard on SPEs • [OTA-BSA] = 0.2 µg/mL • [anti-OTA] = 1:100 • [IgG-HRP] = 1:500 • [IgG-AP] = 1:750 • 1 h competition (10 µL anti-OTA + 90 µL OTA) • IgG-enzyme conjugate incubation: 1 h in milk

  15. 120 100 80 60 % of absorbance 40 20 0 -3 -2 -1 0 1 2 log([OTA] (mg/ml)) Immunosensor Colourimetric OTA detection (SPE) IgG-HRP • IC50 (IgG/HRP) = 7841 µg/L • IC30 (IgG/HRP) = 311 µg/L • IC10 (IgG/HRP) = 12 µg/L • IC50 (IgG/AP) = 2363 µg/L • IC30 (IgG/AP) = 332 µg/L • IC10 (IgG/AP) = 47 µg/L IgG-AP n = 4

  16. 0.05 0 -0.05 -0.10 i (mA) -0.15 -0.20 -0.25 -0.30 -0.35 -0.3 -0.2 -0.1 0 0.1 E (V vs. Ag/AgCl) Immunosensor Electrochemical detection • MPMS(N-methylphenazonium methyl sulfate) Chronoamperometry (single-drop analysis) 2 min substrate incubation Ereading = - 0.2 V for 20 sec MPMS MPMS + HRP + H2O2 0.2 mM MPMS + 20 mM H2O2 in 100 mM acetate buffer with NaCl

  17. 100 80 60 % of absorbance 40 20 0 -3 -2 -1 0 1 2 log([OTA] (mg/ml)) Immunosensor Electrochemical detection MPMS in solution IgG-HRP • IC50 = 1089 µg/L • IC30 = 234 µg/L • IC10 = 51 µg/L n = 3 Total system: 5523 nA (12.6 %) No IgG-HRP: 3509 nA (4.8 %) No anti-OTA: 3500 nA (5.0 %) No H2O2: 3333 nA (7.6 %) No MPMS: 1040 nA (7.1 %)

  18. Conclusions Conclusions • Strategy 1 allows the colourimetric detection of OTA, using wells and SPEs, and the development of an electrochemical immunosensor for the determination of this mycotoxin. • Both IgG-enzyme conjugates, with HRP or AP, can be used. Up to now, good results are observed when working with an electrochemical mediator (MPMS) in solution for the determination of HRP. • However, work is in progress since our aim is to achieve lower limits of detection (0.05-0.1 µg/L).

  19. IMMUNOSENSOROTHER STATEGIES

  20. anti-OTA PAb Enzyme substrate Enzyme product IgG-enzyme conjugate anti-OTA PAb OTA biotin-OTA SAv Screen-printed electrode Future work Strategy 2

  21. Enzyme substrate Enzyme product SAv-enzyme conjugate biotin-OTA OTA anti-OTA PAb Screen-printed electrode Future work Strategy 3

  22. Enzyme substrate Enzyme product OTA-enzyme conjugate OTA anti-OTA PAb Screen-printed electrode Future work Strategy 4

  23. anti-OTA PAb anti-OTA PAb OTA biotin-OTA Av Quartz • Development of an immunosensor for OTA determination based onQCM detection

  24. Development of an immunosensor for OTA determination based on QCM detection Quartz of oscillation frequence of 27 MHz

  25. Streptavidin-Au OTA Av biotin-OTA anti-OTA PAb Gold electrode • Development of an immunosensor for OTA determination based on impedimetric detection

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