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

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

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. Factors Affecting Immobilisation Protein – Surface interactions ? + ? + ? + ? + ? - ? - ? - ? -- δ + δ + δ + δ - δ - δ Non-polar Positive charge Positive Polar Negative charge Negative Polar Non-polarNon-polarNonpolar Hydrophilic Hydrophobic δ - δ Lots of possibilities x Weak, temporary ? + ? + ? + ? + ? - ? - ? - ? -- δ + δ + δ + δ - δ - δ

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

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

13. Plasma Immersion Ion Implantation N+ Insulated wire Nitrogen Plasma -ve biased metal electrode CCCC C C HHHHH H H HHHH H H C H CC C ·C·C HHHHH H H HH H H C·C· H C·C· C·C·

14. 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 OH O C CCCCC CCC C·C· HHHHH H HH HH HHHHH HHH H CC HHH HH C HH H C O C O C·C· C·C·

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

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

17. Results – Surface Groups Surface[C=O] / [C-H] untreated0.0% PIII-treated and exposed to air1.4% PIII-treated not exposed to airunmeasurable copolymer1.5% Soaked in methacrylic acid17.7%

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

19. Results – Immobilised Protein 0% C=0 1.4% 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. Summary Protein immobilisation – Hot topic, potentially revolutionary – Not well understood Hypothesised COOH may be important Results disprove hypothesis – Importance of free radicals? C O OH ·x·x

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

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

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