PAN MECHANIZMY ADSORPCJI BIAŁEKfluid.ippt.pan.pl/seminar/text/Adamczyk-Warszawa-2011.pdf ·...
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Seminarium Mechaniki Płynów IPTT PAN,
Warszawa 2011
J. Haber Institute of Catalysis and Surface Chemistry,
Polish Academy of Sciences,
Cracow, Poland
MECHANIZMY ADSORPCJI BIAŁEK
FAKTY I MITY
Z. Adamczyk
PAN
THE PROBLEMParticles (solutes):
Protein, DNA, viruses, cells
Polyelectrolytes
Colloids
Surfactants
Significance/Processes:
biosensors
separation of DNA,proteins, viruses, cells
immunological assays
affinity chromatography
interfacead
sorp
tion
macroscopic
flow
PREDICTING PARTICLE ATTACHEMENT
KINETICS (RATE AND MONOLAYER COVERAGE)
Particle flux:
jo = - D nb / R
D – diffusion coefficient
nb – concentration in the bulk
R= Rbulk + Rsurf (Qmx)
Rbulk – bulk transport
resistance.
Rsurf - surface resistance
Qmx – from simulations
Su
rfa
ce
Co
ve
rag
e
Adsorption time
IjoI
ta
Monolayer coverageQ
mx
rela
xati
on
tim
e
INTERACTIONS AFFECTING PARTICLE (PROTEIN)
ATTACHEMENT
unknown DLVO bulk
101 102 103 104 h [Ǻ]
10-4 10-3 10-2 10-1 1 H = h/a
102
10
1
1
10
102
Dim
ensio
nle
ss forc
e
att
ractio
n r
ep
uls
ion
_
F
ch
em
ica
l ?
Bo
rn ~
H-7
external H°
Electric double-layer
hydr. shear ~H
hydr. normal ~H 2
dispersion ~H -2
double-layer ~e - a H
hydr. normal
(stagnat. point)
Dimensionless distance
h
a
QUESTION: IS THIS VALID FOR PROTEINS ?
IN VIEW OF ANOMALOUS PHENOMENA SUCH AS:
APPARENT ATTRACTION OF LIKE CHARGES?
TRIPLE REPULSION = ATTRACTION ?
IS THERE SOMETHING WRONG
WITH COULOMB LAW ?
THESIS: BASIC PHYSICS WORKS IN PROTEIN
BUSINESS
APPARENT DEVIATIONS ARE DUE TO WRONG
INTERPRETATION
(LACK OF REFERENCE EXPERIMENTAL RESULTS
FOR MODEL SYSTEMS)
MEANS OF PROVING THIS:
• THEORETICAL CALCULATIONS
AND SIMULATIONS
• EXPERIMENTAL MEASUREMENTS USING in situ
ELECTROKINETIC METHODS
(Streaming potential)
Marian von SMOLUCHOWSKI 1872 –1917
•Statistical physics- fluctuation theory •Brownian motion and diffusion
•Colloid statistics - coagulation theory •Streaming potential-electrokinetic phenomena
WOMEN IN THOSE DAYS …
G.Seurat „La Grande Jatte”
a study in low pixel resolution
G.Seurat „A Sunday on La Grande Jatte”
a study in high pixel resolution
STREAMING POTENTIAL OF BARE SURFACES
Smoluchowski’s formula for homogeneous surfaces (simple shear flows)(Bull. Acad. Sciences de Cracovie 1903, pp 182-199)
e – permittivity, zi – zeta potential of interfaces
Re – electric resistance
For channel flows:
P – hydr. pressure difference
h – dynamic viscosity
r – specific electric resistance
s o i
s s e
I G l
E I R
e z
iζ
Δs i o i
PE Ce rz z
h
sI
STREAMING POTENTIAL OF COVERED SURFACES
Formula for particle (protein) covered surfaces (arbitrary shearing flow)
Fi = correction function describing flow damping
Fp = correction function describing particle induced curent
Particle coverage: (N – surface concentration)2Θ a N
sI
s o i i p pE / C F Θ F Θz z
CORRECTION FUNCTIONS FOR PARTICLE COVERED
SURFACES
(Z. Adamczyk et al. Bull. Pol. Ac. Chem. 1999, 47, 239- 258)
(K. Sadlej, E. Wajnryb, J. Bławzdziewicz,
M.L. Ekiel-Jeżewska, Z. Adamczyk,
J. Chem. Phys. 2009, 130, 144706)
10.2- Θ
iF Θ = e
- 2 6.51Θ
p
6.51 - 2.38Θ 1 1 - eF Θ = Θ
1+ 5.46Θ Θ2
STREAMING POTENTIAL MEASUREMENTS
4
6 3
2
1 3
The streaming potential cell
1 – parallel-plate channel
2 - Ag/AgCl electrodes for streaming potential measurements
3 - electrodes for cell resistance determination
4 - Keithley electrometer
5 - conductivity cell
6 - conductometer
-the parallel plate channel of dimensions
0.027 x 0.29 x 4 cm
- in situ method
Interface with particles
SUBSTRATE SURFACE CHARACTERISTICS Zeta potential of mica vs. pH
M. Wasilewska, Z. Adamczyk, Langmuir, 2011, 27, 689-696
I=10-2M
I=10-3M
3.5 7.4
pH
2 3 4 5 6 7 8 9 10 11 12 13
zp [mV]
-140
-120
-100
-80
-60
-40
-20
0
20
40
60
80
100
A500
S800
10-2M NaCl
10-4M NaCl
10-3M NaCl
CHARACTERISTICS OF MODEL COLLOID PARTICLES (POSITIVE - A500 AND NEGATIVE
S800 LATEX )Z
eta
pote
nti
al
10-2M NaCl
Irreversibly adsorbed particles- Fluctuations
q = 0.1 q = 0.3
COLLOID PARTICLES AT INTERFACES (MICA)
Pair correlation function for latex particles adsorbed on mica
(under the quasi-liquid state)
STRUCTURE OF COLLOID PARTICLE MONOLAYERS(LATEX ON MICA )
Diffusion cell, Points - AFM and optical microscopy the solid lines denote
the RSA model, the dashed line-Langmuir model
Z. Adamczyk et al., Advances CIS, 2010, 153, 1-29
KINETICS OF COLLOID PARTICLE DEPOSITION
IN MODEL SYSTEMS (latex /mica)
I=2x10-5 M I=10-2 M
Q
0.0 0.1 0.2 0.3 0.4 0.5
-0.5
0.0
0.5
1.0
1.5z/zp
REFERENCE DATA FOR COLLOIDAL PARTICLESZETA POTENTIAL OF MICA COVERED
BY POSITIVE LATEX
Points- experimental results: Z. Adamczyk et al., Langmuir , 2010, 26, 9368
lines - exact theoretical results K. Sadlej et al, J. Chem. Phys. 2009, 130, 144706–144711
Red
uce
d z
eta
po
ten
tia
l
Iatex coverage
latex in the bulk
latex on mica
Q
0.0 0.1 0.2 0.3 0.4 0.5
-1.0
-0.5
0.0
0.5
1.0
pH=11.2
pH=11.8
pH=10.1
pH=5.5
zz i
Points- experimental results: Z. Adamczyk et al., Langmuir , 2010, 26, 9368
lines - exact theoretical results K. Sadlej et al, J. Chem. Phys. 2009, 130, 144706–144711
Iatex coverage
Red
uced
zeta
po
ten
tia
l
mica
Latex A500
(negative for pH > 10)
Latex A500
(positive for pH < 10)
mica
REFERENCE DATA FOR COLLOIDAL PARTICLES
ZETA POTENTIAL OF MICA COVERED
BY NEGATIVE AND POSITIVE LATEX
Q
0.35 0.40 0.45 0.50 0.55
zzp
0.0
0.2
0.4
0.6
0.8
1.0
1.2
monolayers of latex particles
bilayers of latex particles
PEI
PAH
ZETA POTENTIAL OF MICA COVEREDBY POSITIVE LATEX PARTICLES
bulk2
1z
bulk
particle covered surface
Red
uce
d z
eta
po
ten
tia
l
Particle coverage
Theoretical
Points- experimental results: Z. Adamczyk et al., Langmuir , 2010, 26, 9368
lines - exact theoretical results K. Sadlej et al, J. Chem. Phys. 2009, 130, 144706–144711
pH
2 3 4 5 6 7 8 9 10 11 12 13
-80
-60
-40
-20
0
20
40
60
80
100z [mV]
.
BULK VS. SURFACE ZETA POTENTIAL OF MICA COVERED BY LATEX PARTICLES
bulk
surface
Zet
a p
ote
nti
al
mic
a/l
ate
x
THE FIBRINOGEN STORY 1. Physicochemical characteristics
Molecular weight [Da] 340 000
Specific density [g·cm-3] 1.38
Specific volume [nm3]
Crystalline state372
Hydrated volume [nm3] 440
Equivalent sphere radius [nm] 4.5
Diffusion coefficient cm2 s-1 2.1x10-7
Hydrodynamic radius [nm] 12 (pH = 3.5, I = 10-2 M)
Molecular shape
(crystalline state)
Approximated shape
bead model A
47.5 nm
THE FIBRINOGEN STORY 2. Electrophoretic mobility and free charge vs. pH
pH4 6 8 10
e [
m
cm
V-1
s-1
]
-2
-1
0
1
2Nc
-40
-30
-20
-10
0
10
20
30
NaOH
TRIS
Ele
ctro
ph
on
etic
mob
ilit
y
Nu
mb
erof
free
charg
es
I=10-2M
M. Wasilewska, Z. Adamczyk, Langmuir, 2011, 27, 689-696
Experimental
M. Wasilewska, Z. Adamczyk, Langmuir,
2011, 27, 689-696
Simulations, RSA, model A
Z. Adamczyk et al., Langmuir,
2010, 26, 11934
THE FIBRINOGEN STORY 3. Fibrinogen monolayers on mica
THE FIBRINOGEN STORY 4. Kinetics of fibrinogen adsorption on mica
diffusion transport, AFM
Bulk transport controlled adsorption !
Z. Adamczyk, M. Nattich, M. Wasilewska, M. Sadowska, JCIS, 2011, 356, 454–464
theory, generalized RSA
experimental points, diffusion cell,
pH=3.5, I=10-3M
N
um
ber
of
mole
cule
s N
/cb
[ m
-2 p
pm
-1]
adsorption time t1/2
[min1/2
]
0 2 4 6 8 10
0
200
400
600
800
THE FIBRINOGEN STORY 5. Zeta potential changes of mica substrate upon
fibrinogen adsorptionFibrynogen I=10-2
, pH=3.5
Qf
0.0 0.1 0.2 0.3 0.4 0.5
z
-80
-60
-40
-20
0
20
40
experimantal
theoretical
Col 24 vs Col 25
Col 27 vs Col 28
Col 30 vs Col 31
Col 22 vs zeta
mica
bulk fibrinogen
mica
bulk fibrinogen
pH=3.5
pH=7.4
Fibrinogen coverage
M. Wasilewska, Z. Adamczyk, Langmuir, 2011, 27, 689-696
Side-on adsorption of fibrinogen !
M. Wasilewska, Z. Adamczyk, Langmuir, 2011, 27, 689-696
MECHANISM OF FIBRINOGEN ADSORPTION ON MICA
DETERMINED BY STREAMING POTENTIAL
Fibinogen coverage Qf
0.0 0.1 0.2 0.3 0.4 0.5 0.6
-1.0
-0.5
0.0
0.5
1.0
side-on
end-on
points,experimental results(microfluidic cell)
theory for side-on adsorption
theory for end-on adsorption
Guy-Chapman model(patchy adsorption)
pH=3.5, I=10-2M
COLLOID ENHANCEMENT OF FIBRINOGEN
MONOLAYERS
Microparticles
Mica substrate Substrate/protein monolayer Replica of protein
monolayer
Step 1
Protein
adsorption
Step 2
Detection
Microparticles: Negative Latex L800 (diameter 800nm)
Positive Latex A800 (diameter 810 nm)
Points: experimental results for negative latex L800, ( ) optical microscopy, ( ) AFM
Lines: theortical results for adsorption site composed of 1 and 2 fibrinogen molecules
Z. Adamczyk, M. Nattich, M. Wasilewska, M. Sadowska, JCIS, 2011, 356, 454–464
LATEX PARTICLE DEPOSITION ON SITES FORMED BY
FIBRINOGEN ON MICA
Fibrinogen coverage Qf
0.0 0.1 0.2 0.3
Neg
ati
ve l
ate
x c
ove
rage
QL/ Q
ma
x
0.0
0.2
0.4
0.6
0.8
1.0
teta latex vs QL/QMAX DLA LINI1
teta latex vs QL/QMAX DLA LINI2
optical microscopy
AFM microscopy
DLVO theory
1
2
Fluctuaction
theory
DLVO
theory
pH=3.5
-- - - - - -
+ +
Points: experimental results for negative latex L800, ( ) optical microscopy, ( ) AFM
Lines: theortical results for adsorption site composed of 1 and 3 fibrinogen molecules
Z. Adamczyk, M. Nattich, M. Wasilewska, M. Sadowska, JCIS, 2011, 356, 454–464
LATEX PARTICLE DEPOSITION ON SITES FORMED
BY FIBRINOGEN ON MICA
Fibrinogen coverage Qf
0.0 0.1 0.2 0.3
Neg
ati
ve l
ate
x c
ove
rag
e Q
L/ Q
max
0.0
0.2
0.4
0.6
0.8
1.0
teta latex vs ql/qmax
teta latex vs ql/qmax dla lini 3
optical microscopy
AFM microscopy
DLVO theory
1
3
pH=7.4-
- - - - -+ +
Fluctuaction
theory
DLVO
theory
Points: experimental results for negative latex L800, by optical microscopy and AFM.
Line: theortical results derived from fluctuation theory.
Z. Adamczyk, M. Nattich, M. Wasilewska, M. Sadowska, JCIS, 2011, 356, 454–464
LATEX PARTICLE DEPOSITION ON FIBRINOGEN
OVERED MICA
Zeta potential z
-60 -40 -20 0 20
Neg
ati
ve
la
tex
co
ve
rag
e Q
L/ Q
max
0.0
0.2
0.4
0.6
0.8
1.0
pH=3.5, I=10-3
pH=3.5, I=10-2
pH=7.4, I=10-2
Col 32 vs Col 31
Fluctuaction
theory
DLVO
theory
-- - - - -+ +
COLLOID ENHANCEMENT OF FIBRINOGEN LAYERS ON MICA(FORMATION OF SUPER-ADSORBING SURFACES ! )
Zeta Potential of Surface z [mV] Zeta Potential of Surface z [mV]
Late
x c
ove
rage
QL
0.0 0.1 0.2 0.3
Fibrinogen coverage Qf
0.0
0.1
0.2
0.3
0.4
0.5
-65 0 20
+
- - - - - -+ +
-- - - - - -
+ +
Fluctuaction
theory
DLVO
theory
Points: experimental results for negative latex L800, by optical microscopy and AFM.
Blue Line: theortical results derived from fluctuation theory.Z. Adamczyk, M. Nattich, M. Wasilewska, M. Sadowska, JCIS, 2011, 356, 454–464
pH=3.5
Henri Rousseau, 1910.
Oil on canvas
L. Szyk-Warszyńska, 2005.
Fluorescein on mica
COLLOIDAL ART
AND REAL...
CONCLUSIONS
• Streaming potential measurements combined with colloid deposition
proved heterogeneous charge distribution over protein molecules.
• This explains anomalous adsorption of fibrinogen at pH = 7.4 and
deviations from predictions of the DLVO theory.
• The Coulomb law is correct, but the application of the continuous
DLVO theory to protein adsorption is wrong.
• Protein adsorption phenomena are governed by ordinary physical laws,
there is no need for introducing additional interactions, especially
the hydrophobic forces
Maria L. EKIEL-JEŻEWSKA
Krzysztof SADLEJ
Eligiusz WAJNRYB
Jakub BARBASZ
Małgorzata NATTICH –RAK
Maria MORGA
Marta SADOWSKA
Monika WASILEWSKA
Maria ZAUCHA
Financial support: Project: N204 0264338
ACKNOWLEDGEMENTS
CRACOW, ART
Beauty at large...
Veit Stoss Altar in Mariacki Church,1477-1489
Lady with the Ermineby Leonardo da Vinci in Czartoryski’s Museum
• Significance of Adsorption (Nanoarchitecture)
• Defining Driving Forces
• Theoretical Methods & Results
• Illustrative Experimental Results
• Conclusions
OUTLINE
Theoretical result for particles covered surfaces
General expression for the reduced zeta potential for particle covered surfaces
ζ the reduced zeta potential of interface with adsorbed particles ζi the zeta potential of the bare interface ζp the zeta potential of particles
Particle coverage
Schematic view of the shear flow past colloid particles adsorbed at a solid/liquid interface.
qqz
zqqz p
i
p
i AA1
:
NSgq
2aSg N the number of particles per unit area
shear flownear interface
ZAGADNIENIE
Detekcja makrocząsteczek na powierzchniach granicznychE0 ~ i0
i0
potencjał (prąd) przepływu
Powierzchnia pokryta centrami (np.antyciałami)
Makrocząstki (antygeny)
i
Powierzchnia pokryta centrami i cząstkami
ΔE ~ Θ