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Creating Surfaces That Immobilize Proteins

Creating Surfaces That Immobilize Proteins. Jenni Tilley Dept. of Materials, Oxford University jennifer.tilley@trinity.ox.ac.uk. Creating Surfaces That Immobilize Proteins. Background What is a Protein? What is Protein Immobilisation? What factors could be affecting immobilisation?

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Creating Surfaces That Immobilize Proteins

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  1. Creating Surfaces That Immobilize Proteins Jenni Tilley Dept. of Materials, Oxford University jennifer.tilley@trinity.ox.ac.uk

  2. Creating Surfaces That Immobilize Proteins • Background • What is a Protein? • What is Protein Immobilisation? • What factors could be affecting immobilisation? • Immobilisation Techniques • What is PIII? • Hypothesis • Results and Interpretation • What does it all mean

  3. O O O O C C N N N C O Protein Primary Structure Peptide bond H H H O O R H H X H C C N N C C H O ? α H C C C O O ? ? α N N C C H H R H H y H H O O n R-group Properties Charged Negative charge Positive charge - δ Polar Non Non Non Non - - - polar polar polar polar Positive Polar δ Negative Polar Other Hydrophilic Hydrophobic

  4. Extended State - Skin Helices – Tendons and Bone H R H C N C O O R H H C N C O Protein Secondary Structure

  5. Protein Tertiary Structure Tertiary Structure Controls Activity

  6. Protein Absorption– Uncontrolled Attachment • E.g. Contact Lenses

  7. Protein Immobilisation – Controlled Attachment • Many varied applications

  8. Applications

  9. Improving the immobilisation • Need to control certain criteria • Control of Protein Activity • Capacity for Protein Loading • Strength of Immobilisation • But…attachment mechanism not understood

  10. - - δ δ + + δ δ + + δ δ - - δ δ - - δ δ - - - - ? ? ? ? + + ? ? + + ? ? + + δ δ - - ? ? + + ? ? - - ? ? + + ? ? Factors Affecting Immobilisation • Protein – Surface interactions Negative charge Positive charge - δ Non Non Non Non - - - polar polar polar polar Positive Polar δ • Lots of possibilitiesx Weak, temporary Negative Polar Hydrophilic Hydrophobic

  11. - - δ δ + + δ δ + + δ δ - - δ δ - - δ δ - - - - ? ? ? ? + + ? ? + + ? ? OH O + + δ δ - - ? ? C + + ? ? - - ? ? + + ? ? Important Surface Characteristics • Difficult to model – trial and error • Two possibilities OR… ·x

  12. - δ + δ + δ - δ - δ - - ? ? + ? + ? + δ - ? + ? - ? + ? Wet Chemistry Technique • Lots of steps • difficult to control • messy! MASK DE-MASK ACTIVATE RINSE RINSE RINSE MODIFY

  13. N+ N+ N+ N+ N+ N+ N+ N+ N+ N+ H H H H H H H H H H H H H H H H H H H H H H H H H H C C C C C C C Plasma Immersion Ion Implantation Nitrogen Plasma C· C· C· ·C C C C Insulated wire -ve biased metal electrode

  14. OH OH OH O O O C C C H H H H H H H H H H H H H H H H H H H H H H H H H H H PIII – what does it do? • Introduces free radicals and, in air, chemical groups • Increases amount of protein attachment • Increases strength of protein attachment • Is this due to the chemical groups or the free radicals? C· C· C· C C C C C C C C C C C C

  15. OH OH OH OH O O O OH O O C C C C C Hypothesis • “Chemical groups are important in the strong immobilisation of protein” • CREATE DIFFERENT SURFACES C· C· C· C· C· C· C·

  16. Infrared spectroscopy C=O vibrations C-H vibrations Methacrylic acid Copolymer

  17. Results – Surface Groups

  18. Results – Immobilised Protein 56.2° 1.4% C=0 90.8° 0% C=0

  19. Results – Immobilised Protein 1.4% C=0 0% C=0 1.5% C=0 18% C=0

  20. Conclusions • PIII offers definite advantages • Air-exclusion makes no difference • Carboxyl groups are not important • Are free radicals important?

  21. OH O C Summary • Protein immobilisation • Hot topic, potentially revolutionary • Not well understood • Hypothesised COOH may be important • Results disprove hypothesis • Importance of free radicals? ·x

  22. - - - δ δ δ + + + δ δ δ + + + δ δ δ - - - δ δ δ - - - δ δ δ - - - - - - ? ? ? ? ? ? + + + ? ? ? + + + ? ? ? OH O + + + δ δ δ - - - ? ? ? C + + + ? ? ? - - - ? ? ? + + + ? ? ? Interpretation – copolymer surfaces HOH HOH HOH HOH HOH HOH HOH HOH

  23. - - - δ δ δ + + + δ δ δ + + + δ δ δ - - - δ δ δ - - - δ δ δ - - - - - - ? ? ? ? ? ? + + + ? ? ? + + + ? ? ? OH OH OH OH OH OH O O O O O O + + + δ δ δ - - - ? ? ? C C C C C C + + + ? ? ? - - - ? ? ? + + + ? ? ? Interpretation – methacrylic surfaces HOH HOH HOH HOH HOH HOH HOH HOH HOH HOH HOH

  24. - - - - δ δ δ δ + + + + δ δ δ δ + + + + δ δ δ δ - - - - δ δ δ δ - - - - δ δ δ δ - - - - - - - - ? ? ? ? ? ? ? ? + + + + ? ? ? ? + + + + ? ? ? ? OH O + + + + δ δ δ δ - - - - ? ? ? ? C + + + + ? ? ? ? - - - - ? ? ? ? + + + + ? ? ? ? Interpretation – PIII-treated surfaces HOH HOH HOH HOH HOH HOH HOH C· C· C· C· C· C· C·

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