Creating Surfaces That Immobilize Proteins

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Creating Surfaces That Immobilize Proteins
Jenni Tilley Dept. of Materials, Oxford
University jennifer.tilley_at_trinity.ox.ac.uk
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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

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Protein Primary Structure
Peptide bond
H
O
H
n
R-group Properties
Charged
Negative charge
Positive charge
-
d
Non
-
polar
Positive Polar
Non
-
polar
Non
-
polar
Non
polar
d
Polar
Negative Polar
Other
Hydrophilic
Hydrophobic
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Protein Secondary Structure
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Protein Tertiary Structure
Tertiary Structure Controls Activity
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Protein Absorption Uncontrolled Attachment

• E.g. Contact Lenses

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Protein Immobilisation Controlled Attachment

• Many varied applications

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Applications
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Improving the immobilisation

• Need to control certain criteria
• Control of Protein Activity
• Capacity for Protein Loading
• Strength of Immobilisation
• Butattachment mechanism not understood

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Factors Affecting Immobilisation

• Protein Surface interactions

• Lots of possibilities x Weak, temporary

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Important Surface Characteristics

• Difficult to model trial and error
• Two possibilities

OR
x
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Wet Chemistry Technique
MASK

• Lots of steps
• difficult to control
• messy!

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Plasma Immersion Ion Implantation
Nitrogen Plasma
Insulated wire
-ve biased metal electrode
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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?

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Hypothesis

• Chemical groups are important in the strong
  immobilisation of protein
• CREATE DIFFERENT SURFACES

C
C
C
C
C
C
C
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Infrared spectroscopy
CO vibrations
C-H vibrations
Methacrylic acid
Copolymer
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Results Surface Groups
Surface CO / C-H
untreated 0.0
PIII-treated and exposed to air 1.4
PIII-treated not exposed to air unmeasurable
copolymer 1.5
Soaked in methacrylic acid 17.7
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Results Immobilised Protein
56.2 1.4 C0
90.8 0 C0
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Results Immobilised Protein
0 C0
1.4 C0
1.5 C0
18 C0
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Conclusions

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

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Summary

• Protein immobilisation
• Hot topic, potentially revolutionary
• Not well understood
• Hypothesised COOH may be important
• Results disprove hypothesis
• Importance of free radicals?

x
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Interpretation copolymer surfaces
HOH
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Interpretation methacrylic surfaces
HOH
HOH
HOH
HOH
HOH
HOH
HOH
HOH
HOH
HOH
HOH
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Interpretation PIII-treated surfaces
HOH
HOH
HOH
HOH
HOH
HOH
HOH
C
C
C
C
C
C
C