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Understanding Carbohydrate Functionality and Developing Novel Microarray and Biosensor Technology

This research explores the significant role of carbohydrates in regulating cell functions and their interactions with proteins. By leveraging collaborations among chemists, biologists, and engineers, we aim to develop innovative microarray and biosensor applications. Key focus areas include characterizing carbohydrate surfaces, understanding their mechanical and chemical properties, and detecting protein interactions using diverse optical methods. This project seeks to enhance our understanding of the extracellular matrix and pave the way for advancements in disease regulation and therapeutic strategies.

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Understanding Carbohydrate Functionality and Developing Novel Microarray and Biosensor Technology

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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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