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Andrew Powell

Carbohydrates and surfaces: Towards understanding cell regulation and developing novel microarray and biosensor applications. Andrew Powell. Carbohydrates: Collaboration and technology development is key. Chemists Analytical Chemists Cell biologists Liverpool, Imperial, Manchester, LJMU

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Andrew Powell

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  1. Carbohydrates and surfaces: Towards understanding cell regulation and developing novel microarray and biosensor applications Andrew Powell

  2. Carbohydrates: Collaboration and technology development is key • Chemists • Analytical Chemists • Cell biologists • Liverpool, Imperial, Manchester, LJMU • UKRC glycoarrays consortium • Imperial, Oxford, Manchester, Liverpool, Dundee, UEA, Cape Town SA. • Surface physicists & Engineers

  3. Cells are covered in carbohydrates • Extracellular matrix, Plasma membrane • Different cells or extracellular matrix – different carbohydrates • Carbohydrates regulate cell function!

  4. Carbohydrates and mechanics • Cartilage: Highly charged = trap water - sponginess • Chemical properties of different carbohydrates/cation forms – spectroscopy • Physical properties of different carbohydrates/ cation forms??

  5. Carbohydrate Family: Heparan sulfatesMultifunctional Protein Regulators Extracellular matrix Immune regulation HS Blood clotting Alzheimer’s, CJD Microbe/ Cancer cell spread Cell growth/development Libraries of different carbohydrate molecules – different abilities? E.g. protein binding/ Cell regulationactivities?

  6. Finding protein-sugar partnershipsTether different carbohydrates to surfaces and see which one a protein interacts with

  7. Finding protein-sugar partnershipsTether different carbohydrates to surfaces and see which one a protein interacts with

  8. Finding protein-sugar partnershipsTether different carbohydrates to surfaces and see which one a protein interacts with Wash

  9. Finding protein-sugar partnershipsTether different carbohydrates to surfaces and see which one a protein interacts with

  10. Finding protein-sugar partnershipsTether different carbohydrates to surfaces and see which one a protein interacts with DETECT

  11. Optical detection methods • Antibodies • Absorbance • Fluorescence Direct • Optical biosensors – refractive index sensors • Polarisation: surface contrast enhancement • Acoustics (tuning fork) – Quartz Crystal Microbalance

  12. Probing many sugars in parallel: microarrays

  13. Probing many sugars in parallel: microarrays Robot: 10-1000s of nl carbohydrate spots on a slide

  14. Probing many sugars in parallel: microarrays nl carbohydrate spots on a slide incubate spots with protein and antibodies Wash & detect spots (carbohydrates) with bound protein using fluorescence scanner • nl per spot:decrease amount of carbohydrate & protein used • 10s-1000s spots: more carbohydrates vs proteins simultaneously

  15. Requirements • Understand how carbohydrate attached to surface? • What is the density & orientation of carbohydrate on surface? • Direct/immediate detection of molecules on surfaces!

  16. Characterisation of molecules on surfaces • Quartz Crystal Microbalance: • Monitoring formation of layers and attachment of sugar on gold chip • Polarisation modulation infra-red adsorption spectroscopy • Characterisation of chemical bonds present on a surface to gain information about layers formed • Dual Polarisation Interferometry (Farfield Analyte): • Monitoring formation of layers and attachment of sugar on aminosilane • Measurement of layer thickness Only do one or a few carbohydrate surfaces at a time Characterise microarrays with lots of carbohydrate spots

  17. Biophysical characterisation of array spots • Optical thickness - SARFAS • Mechanical thickness – AFM • Surface coverage – X-ray photoelectron spectroscopy Monot et al, J. Am. Chem. Soc (2008), 130, 6243-6251

  18. Sarfas: Contrast generated by a film deposited on supports Standard Surf Silicon Standard Surf 25 x better than Si 10 nm thickness film Standard Surf 160 x better than Si 1 nm thickness film http://www.nano-lane.com/

  19. Optical thickness: SARFAS peptide spot/ antibody 2D nano-objects of diameter 2 nm, molecular layers lower than 1 nm http://www.nano-lane.com/ Souplet et al, J. Pept. Sci. (2007), 13, 451-457

  20. ‘See’ lots of spots at once! • 350 nm lateral resolution • Field of view – 60 um to several mm (100s of spots) Monot et al, J. Am. Chem. Soc (2008), 130, 6243-6251

  21. Initial SARFAS data July-Aug: SARFAS equipment loan to test different surface attachments

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