Creating Surfaces That Immobilize Proteins Jenni Tilley Dept. of Materials, Oxford University...
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Transcript of Creating Surfaces That Immobilize Proteins Jenni Tilley Dept. of Materials, Oxford University...
Creating Surfaces That Immobilize Proteins
Jenni TilleyDept. of Materials, Oxford University
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
Protein Primary Structure
n
Peptide bond
Non-polar
Positive charge
Positive Polar
Negative charge
Negative Polar Non-polarNon-polarNon polar
HydrophilicHydrophobic
δ-δ
R-group Properties
Charged
Polar
Other
H
OH
H
N C
O
C?
H
H
NN CC
OO
Cα
H
HR
X
H
N C
O
Ry
H
C?
H
N C
O
H
C?
H
NN CC
OO
H
Cα
H
OOOO
Protein Secondary Structure
H
NC
C
R H
O
O
HR
C N
H
O
Helices – Tendons and Bone
Extended State - Skin
C
Protein Tertiary Structure
Non-polar
Positive charge
Positive Polar
Hydrophilic
Hydrophobic
Negative charge
Negative Polarδ-
δ+
δ+
δ+
δ+
δ+
δ-
δ- δ-
δ-
Represented as:
Non-polar
Positive charge
Positive Polar
Hydrophilic
Hydrophobic
Negative charge
Negative Polarδ-
δ+
Non-polar
Positive charge
Positive Polar
Hydrophilic
Hydrophobic
Negative charge
Negative Polarδ-
δ+
Non-polar
Positive charge
Positive Polar
Hydrophilic
Hydrophobic
Negative charge
Negative Polarδ-
δ+
Positive charge
Positive Polar
Hydrophilic
Hydrophobic
Negative charge
Negative Polarδ-
δ+ Positive Polar
Hydrophilic
Hydrophobic
Negative charge
Negative Polarδ-
δ+
δ+
δ+
δ+
δ+
δ-
δ- δ-
δ-
δ+
δ+
δ+
δ+
δ-
δ- δ-
δ-
Represented as:
Tertiary Structure Controls Activity
Protein Absorption– Uncontrolled Attachment
E.g. Contact Lenses
Protein Immobilisation – Controlled Attachment
Many varied applications
Applications
Improving the immobilisation
Need to control certain criteria
– Control of Protein Activity
– Capacity for Protein Loading
– Strength of Immobilisation
But…attachment mechanism not understood
Factors Affecting Immobilisation
Protein – Surface interactions
?+
?+
?+
?+
?-
?-?-
?- -
δ+δ+
δ+
δ
- δ
- δ
Non-polarPositive charge
Positive Polar
Negative charge
Negative Polar Non-polarNon-polarNonpolar
HydrophilicHydrophobic
δ-δ
Lots of possibilities x Weak, temporary
?+
?+?
+
?+
?-
?- ?
-
?--
δ+
δ+
δ+δ
-δ
-δ
Important Surface Characteristics
Difficult to model – trial and error
Two possibilities
C
O OH
·x
OR…
?+
?+
?+
?+
?-
?-?-
?--
δ+
δ+
δ+
δ
-δ
-δ
?+
?+
?+ ?+
?-
?-
?-
?--
δ+
δ+
δ+
δ
-δ
-δ
Wet Chemistry Technique
Lots of steps
difficult to control
messy!
?+
?+
?+
?+
?-
?-?-
?--
δ+δ
+
δ+
δ
-δ
-δ
MASK ACTIVATE RINSE RINSEDE-MASKRINSEMODIFY
Plasma Immersion Ion Implantation
N+ N+
N+
N+
N+
N+
N+N+
N+
N+
Insulated wire
Nitrogen Plasma
-ve biased metal electrode
C C C C C C
H H H H H H H
H H H H H H
C
H
C C C ·CH H H H H H H
H H H H
C·
H
C· C·
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 C C C C C C C CC
·H H H H H H H H H H
H H H H H HH H HC C
H H H
H HC
H H
H
C
OH
O
C
OH
O
C
·C
·
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
OHO
C
OHO C
OHO
C
OHO
Infrared spectroscopy
Methacrylic acid
Copolymer
C-H vibrations
C=O vibrations
Results – Surface Groups
Surface [C=O] / [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%
Results – Immobilised Protein
90.8°0% C=0
56.2°1.4% C=0
Results – Immobilised Protein
0% C=0 1.4% C=0 1.5% C=0 18% C=0
Conclusions
PIII offers definite advantages
Air-exclusion makes no difference
– Carboxyl groups are not important
Are free radicals important?
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
HOH
HOH
HOH
HOH
HOH HO
H
HOH
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
HOH
HOH
HOH
HOH
HOH
HOH
Interpretation – methacrylic surfaces
C
O OH
?+
?+ ?
+
?+
?-
?-?
-
?--
δ+
δ+
δ+ δ
-δ
-δ
C
O OH
?+
?+
?+
?+
?-
?- ?-
?--
δ+ δ+
δ+
δ
-δ
-δ
C
O OH
?+
?+
?+
?+
?-
?-?-
?--δ+
δ+
δ+
δ- δ
- δ
C
O OH C
O OH C
O OH
HOH
HOH
HOH
HOH
HOH
HOH
HOH
HOH HOH
HOHHOH
Interpretation – PIII-treated surfaces
C
O OH
?+
?+
?+
?+
?-
?-?
-
?--
δ+
δ+
δ+
δ
-δ
-δ
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