creating surfaces that immobilize proteins jenni tilley dept. of materials, oxford university...
TRANSCRIPT
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