Transcript Slajd 1 - Instytut Fizyki PAN

Interaction between glycoproteins and lectins studied using AFM
Kateryna Lebed, Joanna Gryboś, Grażyna Pyka–Fościak, Małgorzata Lekka, Jan Styczeń
The Henryk Niewodniczański Institute of Nuclear Physics, Polish Academy of Sciences,
Radzikowskiego 152, 31-342 Kraków, Poland
1. PROTEIN PATTERNING
Adhesion force measurements
Microcontact printing method as a method for firm protein
anchoring onto a surface without affecting their activity.
CASE a:
(Con A immobilized using polymeric stamp)
60
The unbinding force for
single molecular pair:
F= 191 ± 4 pN
50
events
Fa = 105 ± 2 pN
F= 294 ± 7 pN
40
30
20
F= 400 ± 13 pN
10
0
0,0
0,2
0,4
0,6
0,8
1,0
F adh [nN]
PDMS - polydimethylsilane stamps
CASE b:
(Con A immobilized without patterning)
2. MOTIVATION
The unbinding force for
single molecular pair:
35
F= 221 ± 3 pN
30
The formation of protein arrays onto solid surfaces using
microcontact printing technique as it has many potential
applications including the development of advanced
biosensors.
events
25
F= 330 ± 3 pN
Fb = 108 ± 2 pN
20
15
F= 437 ± 10 pN
10
5
0
0,0
3. ATOMIC
0,2
0,4
0,6
0,8
1,0
F adh [nN]
FORCE MICROSCOPY
The calculated binding forces between concanavalin A
and carboxypeptidase Y was about 100 pN in both cases.
Adhesion maps
Patterned substrate with different chemical domains can
be identified using force spectroscopy:
topography
topography
Force [nN]
1
4. MATERIALS
Interaction between
CaY and ConA
0.20 nN
0.29 nN
0
-200
-100
0
100
Position of sample [nm]
Carboxypeptidase Y (CaY)
adhesion map
5
Force [nN]
Concanavalin A (ConA)
Interaction between
CaY and
glutaraldehyde
adhesion map
0
4.8 nN
The studied protein–carbohydrate interaction was represented by
concanavalin A (Con A) and carboxypeptidase Y (CaY) pair.
Measurements were performed in liquid i.e. TBS buffer
containing 1mM concentrations of Ca++ and Mn++, at room
temperature.
-5
-1000
-750
-500
-250
0
250
Position of sample [nm]
Force distribution
50
ConA – CaY
force
5. RESULTS
F= 219 ± 6 pN
40
PDMS stamps
depth of holes 1.5 µm
width of stripe 5 µm
events
AFM images
30
20
F= 2700 ± 170 pN
ConA – glutaraldehyde
force
10
0
0
depth of holes 1.5 µm
diameter of circle 5 µm
Con A micropatterns
height of
protein layer ≈ 10 nm
width of stripes ≈ 5.1 µm
diameter of circle ≈ 5.1 µm
1
2
3
4
5
F adh [nN]
6. CONCLUSIONS
▪ Protein immobilization using microcontact printing method
was performed with good reproducibility
▪ The calculated unbinding forces between concanavalin A and
carboxypeptidase Y was about 100 pN for both immobilization ways
indicating that microcontact printing technique did not change
biological activity of proteins
▪ Patterned substrate with different chemical domains can be
identified using force spectroscopy