Transcript Au-In and Ni-Sb systems studied by the perturbed angular

The AFM detection of ligand-receptor
interaction on a surface of living cells
Małgorzata Lekka
atomic force microscopy
Position Sensitive
Photodiode
Laser
FN
Cantilever
AFM working in IFJ
FL
Sample
Scanner
Tip
force spectroscopy
 scanner linearization
 spring constant
 A–B signal
 geometry:
 = 10º
 tip – shape
Hoh et al.
scanner linearization
photodiode
laser
 s max 
scanner
n
2  sin 
resonant frequency of a thermally excited cantilever
without protein (conA)
nominal value
 = 7 kHz, k = 0.01 N/m
measured value
 = 5.8 kHz, k = 0.007 N/m
with protein (conA)
0.3 mg/ml
 = 5.8 kHz, k = 0.007 N/m
!
Sader et al.
1 mg/ml
 = 5.4 kHz, k = 0.006 N/m
Fmin  k  k B  T
k = 0.03 N/m
RT
Fmin  11 pN
probe
Standard TGT01
Si3N4
+ APTES (4%) + GL (2.5%)
+ conA (0.3 g/ml)
Si3N4 + APTES (4%)
+ glutaraldehyde (GL; 2.5%)
R = 54 +/- 7 nm
R = 275 +/- 10 nm
number of molecules on probe
 probe size
 protein concentration
 immobilization procedure
Grandbois et al.
Single molecular pair
Moy et al.
rough estimation of the bond strength
k  x2
E
2
bond type
energy
[kJ/mol]
bond length
[nm]
force [nN]
covalent
250
(for S–S)
0.20
4.42
ionic
20
0.25
0.27
Van der Waals
2
0.35
0.02
hydrogen
7
40
0.30
0.30
0.08
0.45
noncovalent
+ hydrophobic forces
Distribution of vitronectin receptors
on a living MC3T3-E1 cell (murine osteoblastic cell)
Kim et al.
AFM tip functionalized
with Helix pomatia
N-acetylgalactosamine in
membrane of group A of RBC
mixed red blood cells
Grandbois et al.
Force [nN]
No of events
Tees et al.
Lee et al.
Eb  10 -20 [J] koff[s-1]
xb [Å]
AGD – αIIbβ3
- 2.67
47.58
1.09
RGD – αIIbβ3
- 2.64
1.53
1.03
ConA-CaY
retraction velocity 3.5 μm/s
No of events
0.3 s
5s
Force [nN]
How to check what it is measured ?
 not functionalized AFM probe
HCV 29 cells
silicon nitride tip
 measurements of known ligand-receptor pair
ConA–PC-3 ConA–ASA ConA–CaY
Force [pN] 116  17
790  32
940  39
blocking of the binding sites
CaY– Con A
free amount of ligand in solution
all or certain number of binding
sites can be blocked
CaY – Con A
+ 1 mg/ml Con A
interaction between
ligand – receptor pair
non-specific
interaction
HCV 29 non–malignant transitional epithelial cells of ureter
different types of interaction
characteristic for cancerous cells
AFM, contact mode
T24 transitional cell cancer of urine bladder
lectins
PHA-L
SNA
ConA
carbohydrates
mannose, glucose
sialic acid
N-acetylglucosamine
binding force
Cell line
Lectin
HCV29
ConA
43.3  3.4
PHA-L
59.9  7.1
T24
Binding force [pN]
SNA
167.2  5.5
ConA
123.6  18.1
PHA-L
152.6  8.2
SNA
76.2  10.9
verification
50 µg/ml ConA
conclusions
 AFM allows detecting molecular interaction on a surface of
living cell
 The spatial arrangement of functional carbohydrate groups on
cell surface was attributed to the density of all types of the
carbohydrate structures (mannose, N-acetylglucosamine, sialic
acids).
 The maximum range of force distribution (presented in
histograms up to 1.2 nN), the size of the adhesion spot (i.e. one
single point on the distribution map ~ 0.95 μm2), the number of
bonds (2–3 for cancerous cells) suggested that ligands present on a
surface of T24 cells formed groups composed of several single
carbohydrate chains involved in adhesion process in the lectin
recognition.
Institute of Medical Biochemistry
Medical College
Jagiellonian University
Piotr Laidler
Joanna Dulińska
Maryla Łabędź
The Henryk Niewodniczański
Institute of Nuclear Physics
Polish Academy of Sciences
Zbigniew Stachura
Małgorzata Lekka
Janusz Lekki
Jan Styczeń
PhD students
Joanna Gryboś
Kateryna Lebed
Grażyna Pyka
Atomic
Force
Microscopy
Histogram
Autocorrelation function
force between single
pair: CaY-ConA
F = 960 +/- 110 pN
C (r ) 
x
max

x
f ( x)  f ( x  r )  dx
bin size
min
large number of data
 = m · F + F0
m = m2
F0 – non-specific force
2


2

2
2
F 
  2  F 

F 
F

f1(F, F2, F0 )·
f2(F, F2, F0 )
more complex interactions
F  const
 F2
single interaction
F  const
 2    F
F · F0