Chem symposium-haddow-oct-2012b

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Seminar - 2012 - UAEU Chemistry Symposium on "Chemistry and Health". Dr. Haddow presentation on Amperometric determination of sialic acid from bio-samples.

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Chem symposium-haddow-oct-2012b

  1. 1. Development of amperometric dual-channel FIA systems for the determination of clinically important free-, bound- and total sialic acid Jody D. Haddow a Sayed A.M. Marzouk a Amr Amin b a Department of Chemistry, United Arab Emirates University, b Department of Biology, United Arab Emirates UniversitySymposium on Chemistry and Health, United Arab Emirates University, Oct, 2012.
  2. 2. OutlineHealth Why sialic acid?Chemistry SA Biosensor Single-channel IER FIA of SA Dual-Channel IER FIA of SA Conclusions Acknowledgements
  3. 3. What is Sialic acid? N-Acteylneuraminic acid (Neu5Ac or NANA) 1 8 9 6 2 7 4 5 3 Bound sialic acid (SAb) Sialic acids are found widely distributed in animal tissues. Important biological roles  43 derivatives of SA but NANA is the most common  Very often the terminal sugar in a glycan  Glycoproteins and Gangliosides
  4. 4. Importance of quantifying SANormal Function – many…- Sialic acid-rich glycoproteins bind lectins – cell adhesion, etc- Cell signaling/recognition, Siglecs – Lectin-Igs Health ImplicationsViral/Bacterial Infection- Influenza viruses bind to sialic acids of the upper respiratory tract.Cancer- Metastatic cancer cells often express a high density of sialic acid-rich glycoproteins.Pharmacodynamics- Epoetin (erythroprotein) used to treat anemia, due to renal failure and cancer chemotherapy.- Baby Formulas- Sialic acid content of the glycan is central of in vitro and in vivo functionality.
  5. 5. Bound sialic acid AmperometricpH sensor (neuraminidase (Sialidase) Biosensor/ IER FIA Free sialic acid Analysis of SA (N-acetyl-neuraminic acid aldolase) A Novel Approach N-Acetyl-D-mannosamine Pyruvate dimethyl-amino- Lactate dehydorgenase, NAD+ Acylglucosamine 2- benzaldehyde Pyruvate oxisdase, O2 epimerase Colorimetric measurements of the N-Acetyl-glucosamine product N-acetylhexosamine H2O2 NADH oxidase aminoantipyrine chlorophenol-4- Peroxidase, p- Amperometric Fluorometric detection measurement of the (present work) generated NADH acetylglucosaminic acid + H2O2 Colorimetric measurements of the produced dye
  6. 6. Anal. Chem 2007, 79 1668-1974Current Research Prototype Amperometric Biosensor for Sialic Acid DeterminationBound-Sialic acid Sayed A.M. Marzouk, S.S. Ashraf, and Khawla A. Al Tayyari Sensors & Actuators B 157 (2011) 647- 653 Sialidase Flow injection determination of sialic acid based on amperometric detection Sayed A.M. Marzouk, Jody D. Haddow, and Amr Amin. Free Sialic acid Free Sialic acid Research Progression Sialic acid Sialic acid aldolase Batch SA biosensor aldolase Flow Injection SA biosensor Pyruvate Pyruvate Pyruvate Flow Injection Enzyme Pyruvate oxidase Reactor oxidase H2O2 H2O2 Dual Channel – Bound and Free IER SA detection
  7. 7. Enzyme strategies sialidase bSA Free Sialic Acid SA Aldolase (SAA) Free Sialic acid Pyruavte N - acetyl D mannosamine 3 PyO Pyruvate PO4 O2 Acetylphosphate CO2 H 2O 2 Pyruvate + free SA + b-SA SD SAA + PYO + SAA Anodic oxidation PYO +H2O2 --> O2 + 2H+ + 2e- PYO Current H2O2 H2O2 H2O2 signal (Py) (Py + SA) (Py + SA +bSA)
  8. 8. SA Amperometric Biosensor – batch mode 14 Stable and Steady-state response 12 Teflon cap 10 Microporous 8 Current, nA PolyEster membrane 6 4 2 0Enzyme layer (face down) Pt disc, 2 mm 0 1000 2000 3000 4000 5000 Time, sec 14 Linear response to SA Kel-F insulating 12 body, 6 mm 10 8 Current, nA 6 4Anal. Chem 2007, 79 1668-1974 2Prototype Amperometric Biosensor for Sialic Acid 0DeterminationSayed A.M. Marzouk, S.S. Ashraf, and Khawla A. Al Tayyari 0 20 40 60 80 100 120 140 160 180 200 Sialic acid Conc, M
  9. 9. SA Amperometric Biosensor – OptimizationsAnal. Chem 2007, 79 1668-1974 1. Buffer Type - PB vs MOPSPrototype Amperometric Biosensorfor Sialic Acid DeterminationSayed A.M. Marzouk, S.S. Ashraf, and 2. TemperatureKhawla A. Al Tayyari 3. Cofactor concentration 4. NANA Aldolase / Py Oxidase ratio 5. Buffer pH 6. % Glutaraldehyde : Total Protein crosslinking ratio (G/T) 7. Enzyme to BSA matrix ratio Helped to lay foundation for current work
  10. 10. Single-Channel Amperometric FIA of SA Sensors & Actuators B 157 (2011) 647- 653A. Biosensor detector Flow injection determination of sialic acid Ref. based on amperometric detection Sayed A.M. Marzouk, Jody d. Haddow, and Amr Amin. electrode Copper lead PolyethyleneFlow electrode body out Teflon flow-cell 15 mm dia Au/Pt/enzyme layer 20 mm dia SS rod with inlet channelPt counter Thermostated Water 13 mm diaelectrode in Copper tube 3 mm Thermostated OD Water out Flow in
  11. 11. Single-Channel Amperometric FIA of SAB. Immobilized Enzyme ReactorSensors & Actuators B 157 (2011) 647- 653Flow injection determination of sialicacid based on amperometric detectionSayed A.M. Marzouk, Jody d. Haddow, and Amr Flow-Amin. through cell IER Water flow out PYO – SAA Copper tube – 10 turns Co-immobilized Water flow in Carrier Pump Injection solution valve
  12. 12. Advantages IER vs Biosensor• Longer operational lifetime which could be due the larger amount of immobilized enzyme• longer residence time which results in almost complete conversion of the substrate• Contrary to biosensors, enzyme immobilization and signal transduction are optimized independently• IEF can be prepared and used by less experienced personnel compared to biosensors Based on these points the SA analysis was further optimized with IER
  13. 13. Amperometric FIA of SA based on an IER – in situ heating Easily controlled and rapid thermostating PYO – SAA Co-immobilized Signal ≈ 3x Reduced stability and linearity!! 5.00 mM 2.00 mM 1.00 mM 0.50mM 0.25 mM 0.1 mMSensors & Actuators B 157 (2011) 647- 653
  14. 14. Amperometric FIA of SA based on an IER - repeatability 15 5.0 mM 13 mm dia Pt electrode 12 23 oC 9 In another experiment, two SA Current, A solutions of 100 and 250 uM were injected (twenty injections 2.0 mM 2.0 mM each) and showed RSD of peak 6 heights of 1.5 and 1.1%, respectively. Data not shown 1.0 mM 1.0 mM 3 0.5 mM 0.5 mM 0.25 mM 0.25 mM 0.1 mM 0.1 mM 0 0 500 1000 1500 2000 2500 3000 Time, secSensors & Actuators B 157 (2011) 647- 653
  15. 15. Construction of the dual-channel Flow Cell RefWorking 1 Working 2 2-CH Potentiostat 2-channel system to allow simple and rapid quantitation of real bio-samples Allow subtraction of a “spy” channel Counter IER-1 IER-2
  16. 16. FIA Systems based on dual IER and Two amperometric detectors 2-CH Potentiostat W1 W2 R1 R2 Split ratio at the Y-connector?  Very stable – primarily controlled by the relative back pressures introduced by the IERs.  Not necessarily 50-50 but each channel is calibrated independently
  17. 17. Relative sensitivities to SA and Py injections – NANA/PyO IER Signal after two 2 mM 1.4 enzymatic conversions 1.2 NANA+PyOCurrent x 10 , A 1.0 Carrier PB pH 7.3, 1.5 mM6 0.8 SA injection T = 37oC 0.6 Sample loop =10 μL 1 mM 0.4 0.75 mM Single channel 0.5 mM 0.25 0.05 0.1 0.2 0.0 12 0 1000 2000 3000 4000 5000 Signal after one Carrier PB pH 7.3, enzymatic conversion 2.0 mM 10 PY injection T = 37oC Current x 10 , A 1.5 mM 86 8X 1 mM Sample loop =10 μL 6 0.75 mM 0.5 mM Single channel 4 0.25 mM 2 0.1 mM 0.05 mM 0 0 1000 2000 3000 4000 5000 Time, s
  18. 18. Relative sensitivities to SA and Py injections – 2-channel 5 1.0 mM Py 4 PY/SA ~ 6 0. 5 mM Current, A Py 3 Channel that must 0.25 mM be normalized and 2 Py subtracted is too intense 1 0 PYO 0. 5 mM 1.0 mM 0.25 mM SA SA SA SAA - PYO1000 2000 3000 4000 Time, s
  19. 19. Further optimization of SA detection in the presence of Pyruvate 2-CH Potentiostat W1 W2R1= SAA - PYOR2= PYO R1 R2 Depletion of pyruvate Catalase PYO PyO = pyruvate + phosphate + O2 acetyl phosphate + CO2 + H2O2 Catalase = 2 H2O2 → 2 H2O + O2
  20. 20. FIA peaks simultaneously obtained for SA and PY Pre-depletion Py/SA = 6 Post-depletion SA/Py = 2.5
  21. 21. Effect of Flow Rate 0.24 1.50 1.75 1.25 1.00FR: mL/min 0.75 Current, A 0.5 0.16 0.08 0.00 BPYO-SAA C PYO 2000 4000 6000 Time, s Balance between sample residence time (in reactor and at electrode) with the rate sample dispersion
  22. 22. Analysis of bound sialic acid – Fetuin Protein 2-CH Potentiostat W1 W2 R1 R2Fetuin GlycoproteinMolecular weight: 48.4 kDaThe composition of bovine fetuin(weight %) is polypeptide 74%, Sialidasehexose 8.3%, hexosamines5.5%, and sialic acid 8.7%. IER his file is in the public domain because it was solely created by NASA. NASA copyright policy states that "NASA material is not protected by copyright unless noted"
  23. 23. FIA peaks simultaneously obtained for SA, Fetuin and PY PYO-SAA PYO
  24. 24. Simultaneous analysis of total SA and PY in simulated serum sample 2-CH Potentiostat W1 W2 R1 R2 Sialidase Catalase PYO Simulated Serum 6% BSA – 140 mM NaCl 10 mg/mL Fetuin 1 mM Py – 2 mM SA
  25. 25. Simultaneous analysis of total SA and PY in simulated serum sample 1.6 6% BSA - 140 mM NaCl 5.0 mM PY 10 mg/mL Fetuin 1.4 1.0 mM PY-2.0 mM SA 1.2 Current x 10 , A 5.0 mM SA 5.0 mM SA 1.06 5.0 mM 0.8 PY 0.6 2.0 mM SA 2.0 mM 1.0 mM SA 0.4 1.0 mM SA 1.0 mM PY PY 0.2 0.0 1.8 0 1000 2000 3000 4000 1.6 FR = 2.8 mL/min FR = 1.5 mL/min FR = 2.8 mL/min 1.4Current x 10 , A 1.26 1.0  PY signal diminished at reduced flow rate: More time for removal 0.8  bSA was completely hydrolyzed at the high FR 0.6 0.4 0.2 0.0 0 1000 2000 3000 4000 Time, s
  26. 26. Conclusions The problem of intrinsic high sensitivity towards pyruvate was resolved using PYO-catalase sequence. The split ratio was stable as indicated by the calibration stability. The flow cell design proved excellent to provide fast, sensitive and reproducible response. The first simultaneous FIA analysis of PY, SA and or b-SA was successfully demonstrated. The reliability of the analytical systems was evaluated by analyzing PY, SA and bSA in simulated serum sample
  27. 27. Acknowledgements UAEU for the financial support Prof. Sayed Marzouk and Dr. Amr Amin for a fruitful collaboration Khawla A. Al Tayyari early optimization of biosensor Thank-you
  28. 28. Formation of the protective polymeric layerCyclic voltammograms obtained for electropolymerization of 1,3-diaminobenzne(m-phenylenediamine) at two simultaneous Pt disc electrodes Electrode 1 ELectrode 2 200 200 150 150Current x 10 , A6 100 100 50 50 0 0 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Potential (E), V vs SCE Potential (E), V vs SCE Tested against oxidizable species: Thiamine pyrophosphate (TPP), acetaminophen (4- acetamidophenol) (AAP), and uric acid (UA) – blocked by polymer- data not shown
  29. 29. Signal Stability/Repeatability (2-ch split flow) 1 mM SA – 1.5 mL/min – 100 µL injection 0.18 Current, A 0.09 0.00 SAA - PYO B PYO C 500 1000 1500 2000 2500 Time, s- no fluctuation in split ratio- actual ratio not critical - channels calibrated independently
  30. 30. (A) Sample Future Work Waste in Analyzing biological samples. Serum, breast milk, formula, etc. Single channel response Expanding the current study to more comprehensive multi-channel analysis. Time Immobilized enzyme reactors (B) Sample Waste in Ch-1 Ch-2 Ch-3 Ch-4 Ch-5 n channel response Time Ch-1 Ch-2 Ch-3 Ch-4 Ch-5

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