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)

ORIGIN OF STREAMING POTENTIAL

Marian von SMOLUCHOWSKI 1872 –1917

•Statistical physics- fluctuation theory •Brownian motion and diffusion

•Colloid statistics - coagulation theory •Streaming potential-electrokinetic phenomena

SMOLUCHOWSKI’S EXPRESSIONS

FOR ELECTROPHORETIC MOBILITY

AND STREAMING POTENTIAL

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

OTHER MEASUREMENTS

AFM - INTEGRA

Dynamic viscosity

measurements

Dynamic light scattering

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)

V. van Gogh “The Starry Night”

FLUCTUATIONS IN THE SKY

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

HOW ABOUT REAL LIFE?

THE FIBRINOGEN STORY BEGINS

THE FIBRINOGEN MOLECULE

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

Photo by J.Barbasz

Cracow, Main Market Square

THANKS FOR ATTENTION

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

mV50z

positive latex

d = 0.43 m

mV50z

negative latex

d = 0.91 m

MONODYSPERSYJNE

SUSPENSJE

KOLOIDALNE