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Journal of Colloid and Interface Science 356 (2011) 454–464

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Journal of Colloid and Interface Science


www.elsevier.com/locate/jcis

Deposition of colloid particles on protein layers: Fibrinogen on mica


Z. Adamczyk ⇑, M. Nattich, M. Wasilewska, M. Sadowska
Institute of Catalysis and Surface Chemistry, Polish Academy of Science, Niezapominajek 8, 30-239 Cracow, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Colloid particle deposition was applied to characterize fibrinogen (Fb) monolayers on mica, which were
Received 21 October 2010 produced by controlled adsorption under diffusion transport. By adjusting the time of adsorption and the
Accepted 3 January 2011 bulk Fb concentration, monolayers of desired surface concentration were obtained. The surface concen-
Available online 14 January 2011
tration of Fb was determined directly by AFM enumeration of single molecules adsorbed over the sub-
strate surface. It was proven that Fb adsorbed irreversibly on mica both at pH 3.5 and at pH 7.4 with
Keywords: the rate governed by bulk transport. The electrokinetic properties of Fb monolayers produced in this
Colloid particle deposition
way were studied using the streaming potential method. The dependence of the apparent zeta potential
DLVO theory
Fibrinogen layers on mica
of Fb monolayers was determined as a function of the coverage. It was shown that for pH 3.5 the initial
Heterogeneous surface deposition negative zeta potential of the mica substrate was converted to positive for Fb coverage exceeding 0.16. On
Streaming potential measurements the other hand, for pH 7.4, the zeta potential of a Fb-covered mica remained negative for the entire cov-
Zeta potential of fibrinogen-covered mica erage range. The charge distribution in Fb monolayers was additionally studied using the colloid deposi-
tion method, in which negatively and positively charged polystyrene latex particles (ca. 800 nm in
diameter) were used. An anomalous deposition of negative latex particles on substrates exhibiting a neg-
ative zeta potential was observed. Results of these experiments were quantitatively interpreted in terms
of the fluctuation theory assuming that adsorption sites consisted of two and three Fb molecules, for pH
3.5 and 7.4, respectively. These results suggested that for pH 7.4, the distribution of charge on Fb mole-
cules was heterogeneous, characterized by the presence of positive patches, whereas the average zeta
potential was negative, equal to 19 mV. The utility of the colloid deposition method for studying Fb
monolayers was further demonstrated in deposition experiments involving positive latex particles. It
was shown that for a rather broad range of fibrinogen coverage, both the positive and the negative latex
particles can adsorb on surfaces covered by Fb, which behaved, therefore, as superadsorbing surfaces. It
was also concluded that the colloid deposition method can be used to determine the Fb bulk concentra-
tion for the range inaccessible for other methods.
Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction using electron microscopy. Recently, many atomic force micros-


copy (AFM) studies were conducted with the aim of determining
Adsorption of proteins at solid interfaces is important in many conformations of fibrinogen on hydrophilic and hydrophobic solid
fields of science and in various practical processes. It is involved substrates [5–12].
in thrombosis, artificial organ failure, plaque formation, the fouling Experimental studies on the kinetics of fibrinogen adsorption
of contact lenses and heat exchangers, in ultrafiltration and mem- are also abundant. Malmsten, using ellipsometry, determined the
brane filtration, where it exerts a negative influence. kinetics of fibrinogen adsorption on methylated silica for physio-
On the other hand, controlled protein deposition on various sur- logic conditions, i.e., pH 7.4 and I = 0.15 M [13].
faces is a prerequisite of their efficient separation by chromatogra- Systematic measurements of fibrinogen deposition were per-
phy, filtration, for biosensing, bioreactors, immunologic assays, etc. formed by Ortega-Vinuesa et al. [5,6], who measured the thickness
Numerous studies were devoted to fibrinogen (Fb), because of of the protein layer on silicon plates by ellipsometry as a function of
its fundamental role in blood clotting, platelet adhesion, thrombo- pH and ionic strength, for a fibrinogen bulk concentration of
sis, angiogenesis, wound healing, and tumor growth. 70 ppm.
Fibrinogen size, shape, and conformations on surfaces (mostly Precise kinetic measurements of fibrinogen adsorption on sili-
mica) were determined by Hall and Slayter [1] and others [2–4] con and modified glass surfaces forming parallel-plate channels
were performed using the in situ fluorescent TIRF technique by
Santore and co-workers [11,14,15]. These results were supple-
⇑ Corresponding author. Fax: +48 12 425 19 23. mented with an interesting AFM determination of the kinetics of
E-mail address: ncadamcz@cyf-kr.edu.pl (Z. Adamczyk). fibrinogen adsorption for a low coverage range.

0021-9797/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jcis.2011.01.009
Z. Adamczyk et al. / Journal of Colloid and Interface Science 356 (2011) 454–464 455

A significant spread in the maximum coverage of fibrinogen and the amount of adsorbed colloid, which can be assessed quan-
was reported in these works with the lowest and highest value titatively via microscopic counting. Using the colloid deposition
ranging between 1.4 and 11 mg m2. It seems that this discrepancy method, in conjunction with electrokinetic characteristics of pro-
can be attributed to different adsorption mechanisms of fibrinogen tein-covered substrates, one can determine mechanisms of colloid
on various substrates and for various concentration regimes. For deposition on heterogeneous surfaces, which is the primary goal of
hydrophilic surfaces, and the low concentration regime, fibrinogen this work.
adsorption occurs according to the side-on mechanism with the As shown previously [14,24–27], in the case of heterogeneous
maximum amount of adsorbed protein equal to 1.4–1.7 mg m2, surfaces the standard DLVO theory is not applicable because the
depending on the degree of hydration. This is in accordance with net kinetics of colloid particle deposition is governed by favorable
recent theoretic results discussed in Ref. [16]. areas (patches), whose zeta potential deviates from the average va-
For hydrophobic surfaces and higher fibrinogen concentrations, lue pertinent to the entire substrate surface. In view of these pre-
the end-on adsorption of the protein becomes feasible with the dictions, another goal of our work is to determine the range of
maximum coverage many times higher than for the side-on config- applicability of this theory for predicting deposition kinetics of col-
uration. However, since the binding energy in the end-on configu- loid particles on heterogeneous surfaces produced by controlled
ration is much lower than for the side-on orientation, a partial fibrinogen adsorption.
reversibility of adsorption occurs, which can explain the source The research presented in this work has also a practical impact
of discrepancies. since the colloid deposition method can be exploited for a quanti-
Contrary to kinetic aspects, little is known about the electrostat- tative determination of protein coverage and consequently for
ics of fibrinogen monolayers on solid substrates under wet condi- detecting the bulk protein concentration.
tions, which is of vital significance. This kind of information can be We focus our attention on fibrinogen whose bulk characteristics
derived from electrokinetic studies, e.g., streaming potential mea- and surface electrokinetic properties have been precisely deter-
surements performed previously for lysosyme [17,18], bovine ser- mined, which enables a quantitative analysis of the experimental
um albumin (BSA), immuno-gamma globulins IgG [19] and for data.
chymotrypsin [20]. Interestingly, a significant protein adsorption
was observed in the latter work despite the fact that both the mica
2. Materials and methods
substrate and the protein molecules were negatively charged. This
effect was explained by a dipolar distribution of the charge on the
2.1. Materials
protein.
There are only few works devoted to electrokinetic measure-
Fibrinogen from bovine plasma, fraction I, type IV (65% protein,
ments of fibrinogen adsorption on solid substrates. Zembala and
containing approximately 20% sodium chloride and 15% sodium
Dejardin [21] determined variations in the streaming potential of
citrate) was purchased from Sigma (F4753) and used without fur-
silica capillaries on fibrinogen adsorption as a function of time
ther purification. Protein was 93% clottable.
for pH 7.3 and the ionic strength of 102 M. A systematic increase
Ruby muscovite mica obtained from Continental Trade was
in the negative potential was observed on fibrinogen adsorption,
used as substrate, and was freshly cleaved immediately prior to
which was also negatively charged under these conditions.
use without any pretreatment.
Similar studies were performed in the work noted above by Kal-
Water was purified using a Millipore Elix 5 apparatus. Chemi-
asin and Santore [15] who determined the zeta potential of nega-
cals and reagents (Trizma base, sodium chloride, hydrochloric acid,
tively charged silica spheres (1 lm of diameter) covered by a
sodium hydroxide) were commercial products of Sigma–Aldrich
controlled amount of fibrinogen using microelectrophoresis. A
and used without further purification.
monotonic increase in the negative zeta potential of silica particles
Positively charged amidine polystyrene latex (Invitrogen) and
was observed on fibrinogen adsorption, which attained a limiting
negatively charged sulfonate polystyrene latex (synthesized in
value of 20 mV for fibrinogen coverage larger than 1 mg m2
our laboratory) were used in this work.
(for the ionic strength of 5–26 mM, NaCl).
Recently, systematic studies of fibrinogen adsorption on mica,
monitored by AFM and streaming potential measurements, were 2.2. Experimental methods
reported [22]. The dependence of the apparent zeta potential of
mica on the coverage of fibrinogen was determined. A quantitative The solutions of Fb used in subsequent measurements were
interpretation of these experimental data was achieved in terms of prepared as follows: a known amount of protein powder was dis-
a theoretic model considering a 3D adsorption of fibrinogen mole- solved in NaCl aqueous solution having an ionic strength of
cules as discrete particles, approximated as strings of touching 1  103 or 1  102 M. The pH of these Fb solutions was regulated
beads. The Gouy–Chapman model, based on the concept of a con- by the addition of HCl or NaOH. Then, the suspension was gently
tinuous charge distribution, was proven to be inadequate. It was mixed using a magnetic stirrer, and filtered through the Millex-
also suggested that anomalous adsorption occurring at pH 7.4 GS 0.45-lm filter to eliminate aggregates and impurities. The
could be explained in terms of a heterogeneous charge distribution experimental temperature was kept constant at 293 ± 0.1 K.
on fibrinogen, whose molecules exhibited patches of positive The true concentration of Fb in these solutions was determined
charge, even if the net charge was negative. using a high precision densitometer (Anton Paar, type DMA5000 M)
Although the streaming potential method offers the unique pos- by measuring the specific density of the fibrinogen solutions (nom-
sibility of direct, in situ measurements, it is tedious, and does not inal Fb content of 200–2000 ppm) and the specific density of the
provide one with information on the local distribution of coverage supernatant solution acquired by a membrane filtration.
and charge within the protein monolayer. These concentrated stock Fb solutions were then diluted prior
Therefore, in this work, a complementary method of analyzing to the adsorption experiments to a desired degree (usually 0.5–
protein layers on solid substrates under wet, in situ conditions is 5 ppm).
used. The technique, referred sometimes to as the colloid enhance- The purity of the Fb solutions was checked by the dynamic sur-
ment (CE) [23], consists in unspecific deposition of larger colloid face tension measurements carried out using the pendant drop
particles onto protein monolayers. This enables one to determine shape method. There was practically no change in the surface ten-
a functional relationship between the amount of adsorbed protein sion of the 10 ppm solution of Fb within a 2-h time period, which is
456 Z. Adamczyk et al. / Journal of Colloid and Interface Science 356 (2011) 454–464

much longer than the typical time of DLS and deposition measure- particles considered was ca. 2000, which ensured that these mea-
ments, which usually last 15–60 min. surements had a relative precision below 2%, as determined by var-
The diffusion coefficient of Fb under various conditions was iance analysis. It is worthwhile noting that the coverage was
determined by dynamic light scattering (DLS), using the Zetasizer determined in absolute terms, i.e., H ¼ Sg hNi, where hNi is the
Nano ZS Malvern instrument (measurement range of 0.6 nm– average surface concentration of particles (number of particles
6 lm). This technique measures the time-dependent fluctuations per unit area) and Sg ¼ pa2 is the cross section of the particle (a
in the intensity of scattered light which occur because particles un- is the particle radius). Consequently, the coverage represents the
dergo Brownian motion. Analysis of these intensity fluctuations fraction of the entire surface area occupied by particles.
enables determination of the diffusion coefficients of particles However, because of multiple scattering, the optical microscope
which are converted into size distributions. method became less accurate for coverage exceeding 0.3. In this
The microelectrophoretic mobility le of Fb was measured using case the AFM method was used under dry conditions to determine
the Zetasizer Nano ZS Malvern instrument deploying the laser the surface concentration of particles and their coverage.
Doppler velocimetry (LDV) technique (measurement range of
3 nm–10 lm). The zeta potential was calculated using the equation
3. Results and discussion
3g
f¼ l; ð1Þ The physicochemical characteristics of Fb, latex particles, and
2eFðjaÞ e
the mica substrate, needed for a quantitative interpretation of
fibrinogen adsorption and latex deposition data, were initially
where f is the zeta potential of proteins, g is the dynamic viscosity
determined. These comprised the diffusion coefficient of Fb
of water, e is the dielectric constant of water, and F(ja) the function
(hydrodynamic diameter), microelectrophoretic mobility, and
of the dimensionless parameter ja = a/Le, Le ¼ ðekT=2e2 IÞ1=2 is the
electrokinetic (zeta) potential.
double-layer thickness, e the elementary charge, P k2 the Boltzman It was found by DLS measurements that the diffusion coefficient
constant, T the absolute temperature, I ¼ 12 i c i zi is the ionic
of Fb was independent of its bulk concentration (within the range
strength, ci the ion concentrations, zi is the ion valency, and a the
of 100–2000 ppm), assuming an average value of 2.1  107
characteristic dimension of a particle or a protein molecule (e.g.,
cm2 s1 for the ionic strength range of 103–102 M and for pH
its hydrodynamic radius).
3.5 and 7.4. Using this value the Stokes hydrodynamic radius of
AFM measurements of Fb layers were carried out using the NT-
Fb was calculated using the known dependence [32].
MDT Solver PRO device with the SMENA SFC050L scanning head.
The measurements were performed in semicontact mode using a kT
silicon probe (polysilicon cantilevers with resonance frequencies RH ¼ ; ð2Þ
6pgD
of 240 kHz +/10% or 140 kHz +/10%, a typical tip curvature of
10 nm, and a cone angle less than 20°). The protein solution of a where RH is the hydrodynamic radius, and D is the diffusion
desired concentration was allowed to adsorb on the surface for coefficient.
an appropriate time interval. The substrate was then removed, It was calculated from Eq. (2), that RH = 12.1 nm (see Table 1). It
rinsed for 30 s using double distilled water, and dried under a gen- is interesting to observe that this is much higher than the radius of
tle nitrogen stream. the equivalent sphere equal to 4.5 nm [32] and reflects an elon-
The latex particle size, as a function of ionic strength and pH, gated shape of Fb determined from crystallographic data (see
was determined using a laser diffractometer (LS 13 320 particle Table 1).
size analyzer, Beckman Coulter) with an accuracy of a few percent The electrophoretic mobility of fibrinogen as a function of pH
and independently by the DLS method using the Malvern Zetasizer and the ionic strength was also determined according to the meth-
Nano ZS. od described above. Knowing the electrophoretic mobility of a par-
The zeta potential of the latex was determined as a function of ticle, one can calculate the average number of uncompensated
the ionic strength and pH using the ZetaPals Brookhaven and the electrokinetic charges per molecule Nc from the Lorenz–Stokes
Zetasizer Nano ZS. relationship [32] and the zeta potential of fibrinogen ff from Eq.
Streaming potential measurements were carried out using a (1). It was determined that ff was positive, equal to 24 and
home-made apparatus, exploiting the parallel-plate channel flow, 28 mV for pH 3.5 and the ionic strength of 102 and 103 M,
according to the procedure described previously [22,28–30]. respectively (see Table 1). On the other hand, for pH 7.4, ff became
Fibrinogen adsorption was carried out in a diffusion cell of a negative, equal to 19 and 21 mV for the ionic strength of 102
volume equal to 40 ml, filled with a protein suspension of a desired and 103 M, respectively (see Table 1). From the electrophoretic
concentration, ionic strength, and pH. Freshly cleaved mica sheets mobility measurements, it was also determined that the isoelectric
(usually 4–8) were placed vertically in the cell for a desired time. point of fibrinogen (zero value of its zeta potential) was pH 5.8.
Afterward, some of the mica sheets (2–4) were rinsed with a The size of the latex particles was determined by laser diffrac-
stream of pure buffer solution for 30 s and dried under a gentle tometry and the DLS method. Both values fell within the experi-
stream of nitrogen. Afterward, the number concentration of fibrin- mental margins of error estimated to be 5%. Thus, the average
ogen was directly determined by AFM examination. diameter of the A800 (positive) latex for the pH range 3–7.4 was
The rest of the mica sheets were used in latex deposition exper- 810 nm (ionic strength 103–102 M) and for the L800 (negative)
iments performed in another diffusion cell, having the dimensions latex 800 nm, for the same range of pH and ionic strength (see
of 2  2  5 cm. It was filled with the latex suspension, typically Table 1). The size and shape of latex particles were also checked
having the number concentration of 1–2  1010 cm3. Wet mica by electron microscopy (SEM); see Fig. 1. The average diameter
sheets, precovered by Fb monolayers as described above, were of the A800 latex (determined from ca. 200 particles) was practi-
then immersed vertically into the latex suspension. Latex article cally the same as that determined by the laser diffractometer.
deposition proceeded over a desired time. The true coverage of The zeta potential of both latex particles was also determined as
particles was determined by a direct optical microscope counting a function of pH and ionic strength. As can be seen (see Table 1),
under wet conditions, as described in our previous works the A800 zeta potential was fairly independent of pH, assuming
[28–31]. Particles were counted over 10–20 equal sized areas cho- 77 mV for I = 103 M and 95 mV for I = 102 M. The zeta potential
sen randomly on the mica sheet. The net number of deposited of the L800 latex was also practically independent of pH assuming
Z. Adamczyk et al. / Journal of Colloid and Interface Science 356 (2011) 454–464 457

Table 1
Physicochemical properties of particles and the mica substrate.

Substance Property
Mica Latex A 800 Latex L 800 Fibrinogen
Zeta potential f (mV) pH 3.5
103 M 63 77 82 28
102 M 52 95 102 24
Zeta potential f (mV) pH 7.4
103 M 112 75 80 21
102 M 80 95 105 19
2 RH (nm) averaged I = 102–103 M – 810 800 24.2
47 nm

Fibrinogen molecular shape

7nm

Fig. 1. Latex particles on mica, SEM images, (a) the L800 latex, (b), the A800 latex.

an average value of 82 mV (for I = 103 M) and 102 mV for


I = 102 M.
The zeta potential of the mica substrate was determined via the
streaming potential measurements according to the procedure de-
scribed elsewhere [22,28–30]. For the ionic strength of 103 M, the
zeta potential of mica was 63 mV for pH 3.5 and 112 mV at pH
7.4. For the ionic strength of 102 M, the zeta potential of mica was Fig. 2. Fibrinogen monolayers on mica (deposition conditions: cb = 0.3 ppm, AFM
less negative, equal to 52 mV for pH 3.5 and 80 mV for pH = 7.4 images, semicontact mode, air). (a) Surface concentration of fibrinogen
(see Table 1). N = 125 lm2 (pH 3.5). (b) Surface concentration of fibrinogen N = 123 lm2 (pH
These results suggest that Fb should adsorb on mica for pH 3.5, 7.4).
due to favorable electrostatic interactions, because the zeta poten-
tial of Fb is positive and the zeta potential of mica strongly nega- atic Fb adsorption experiments were conducted under diffusion-
tive. For pH 7.4, both the Fb and the mica zeta potentials were controlled transport, according to the procedure described above.
negative, which is thought to prevent Fb adsorption because of The number of adsorbed Fb molecules on the mica substrate im-
electrostatic repulsion. In order to verify this hypothesis, system- mersed in the Fb suspension was determined by a direct AFM
458 Z. Adamczyk et al. / Journal of Colloid and Interface Science 356 (2011) 454–464

counting. The topology of fibrinogen monolayers on mica deter- The results shown in Fig. 3 have interesting practical implica-
mined this way is shown in Fig. 2 for pH 3.5 and 7.4. As can be seen, tions showing that one can determine in a quite precise and conve-
fibrinogen molecules appear as isolated entities, which facilitates nient way the bulk concentration of Fb if it is unknown. This can be
their enumeration by AFM. Therefore, using this counting proce- done by plotting N determined by AFM vs. the square root of
dure the kinetics of Fb adsorption can be determined. adsorption time, t1/2. Knowing the slope of this dependence
It is interesting to note that a similar AFM counting procedure of sf ¼ DN=Dt1=2 , one can determine the concentration of fibrinogen
isolated fibrinogen molecules was used by Tsapikouni and Missirlis in the bulk (in ppm) from the simple relationship.
[12], who determined its coverage on mica for pH 7.4 and ionic  p 1=2
Mw
strength of 1.5  102, 0.15, and 0.5 M. cb ¼  1013 sf ð4Þ
Av 2:4D
In Fig. 3, the kinetics is shown as the dependence of the reduced
surface concentration of Fb (number of Fb molecules per lm2 for where cb is expressed in ppm and sf is expressed in lm2 min1/2.
unit bulk concentration of 1 ppm) N/cb on the square root of the Using the value of the diffusion coefficient and molecular
adsorption time t1/2 (I = 103 M, pH 3.5, and 7.4, T = 293 K). As weight pertinent to fibrinogen one can express Eq. (4) in the simple
can be seen, the experimental data in Fig. 3 are agreement with form
theoretic results (depicted by the solid line) stemming from the
C b ¼ 0:014sf : ð5Þ
formula, pertinent to an irreversible, diffusion-controlled adsorp-
tion mechanism [33,34], From Eq. (5) one can directly determine the bulk concentration
 1=2 of Fb even below 0.1 ppm, which is rather difficult using other
D methods.
N¼2 t1=2 nb ð3Þ
p Knowledge of the surface concentration of Fb is necessary for a
proper interpretation of results for colloid deposition
where nb = Cb Av  106/Mw is the number concentration of Fb mol- measurements.
ecules in the bulk (Cb is the Fb concentration in ppm, and Av is the Other data needed for this purpose are the zeta potential f of
Avogadro constant). mica covered by Fb monolayers, which were acquired via stream-
This suggests that Fb adsorption on mica for pH 3.5 was irre- ing potential measurements. It is to be emphasized that our
versible and bulk transport controlled with negligible surface streaming potential measurements were carried out under wet,
transport resistance. This seems quite natural considering the in situ, conditions, which excludes any conformation changes of
strong attraction between the Fb molecules and the oppositely fibrinogen monolayers induced by drying.
charged mica substrate. Results are presented in Fig. 4 showing the dependence of f on
However, similar Fb adsorption kinetics was also observed in the coverage of Fb, calculated from the equation
the case of pH 7.4 (see Fig. 3), where a strong electrostatic repul-
sion was expected due to the same sign of zeta potential of Fb H ¼ Sg N; ð6Þ
and mica. Such anomalous fibrinogen deposition under physiologic where Sg is the characteristic cross section of the protein molecule.
conditions (pH 7.4) was also previously observed by Zembala and As demonstrated in Refs. [16,22] the characteristic cross-
Dejardin [21], Malmsten [13], Ortega-Vinuesa et al. [5,6], Toscano sectional Sg for the side-on adsorption of Fb on mica is equal to
and Santore [11] and Kalasin and Santore [15]. 128 nm2. Using the characteristic cross section for the side-on
As suggested in Refs. [9,15], the attractive interactions at this adsorption, the jamming coverage for fibrinogen becomes 0.29
pH may originate from the heterogeneous charge distribution over [16].
the fibrinogen molecule. Hence, besides being negatively charged, It can be seen in Fig. 4a and b that for pH 3.5, where Fb is pos-
the Fb molecule exhibits positively charged patches, which can itively charged, a steep, quasilinear increase in the zeta potential of
form stable electrostatic bonds with the mica surface. This effect mica is observed for Hf < 0.1. Then, for Hf > 0.16, the zeta potential
was confirmed in a recent publication [22]. became positive and approached saturation values, which were
markedly lower than the bulk values of the zeta potential of fibrin-
800
ogen (equal to 28 and 24 mV for 103 and 102 M, NaCl, respec-
tively). It is interesting to note that the f on Hf dependencies
shown in Fig. 4 are quite similar for both ionic strengths, i.e.,
103 and 102 M. They can be adequately reflected in terms of
600 the theoretic model (depicted by solid lines in this figure) postulat-
N/cb [µm ppm ]
-1

ing a irreversible, side-on adsorption of Fb [16,22]. In this theoretic


approach, a 3D distribution of charge over Fb molecules is consid-
-2

ered, as well as the flow damping effect due to adsorbed molecules


400
[35,36]. It is interesting to note that this model was successfully
applied before for interpretation of zeta potential of surfaces cov-
ered by colloid particles and polyelectrolytes [28–31,37,38]. An
200 interesting feature, which was confirmed in these experiments is
that the limiting value of the zeta potential of particle-covered sur-
faces is 0.71 fp (where fp is the bulk zeta potential of the particle or
protein molecule) rather than fp as intuitively expected.
0
0 2 4 6 8 10 Analogous zeta potential data obtained for pH 7.4 are shown in
t1/2 [min1/2] Fig. 4c. (I = 102 M). As can be seen, no inversion of the mica zeta
potential was observed, because the bare mica zeta potential and
Fig. 3. The dependence of the reduced surface concentration of fibrinogen N/cb the bulk zeta potential of Fb were both negative. However, in this
[lm2 ppm1] on the square of adsorption time t1/2, I = 103 M. The solid points case, theoretic results calculated by assuming a uniform charge
denote experimental results obtained by a direct AFM enumeration for pH 3.5
(various bulk concentrations of fibrinogen) and the hollow points denote results
distribution over fibrinogen molecules corresponding to the bulk
obtained for pH 7.4. The solid line shows exact theoretic results obtained using the value of ff = 19 mV (solid line in Fig. 4c) deviate from experimen-
RSA model. tal points, which lie below. This suggests that the effective zeta
Z. Adamczyk et al. / Journal of Colloid and Interface Science 356 (2011) 454–464 459

(a) 40 itively charged patches on the fibrinogen molecule, which could be


responsible for the favorable electrostatic interactions with mica.
20
Because of the presence of positive patches, the local zeta potential
of negative patches, exposed to the liquid, becomes more negative,
than the average bulk value.
0
In order to check this prediction, latex deposition experiments
were performed according to the procedure described above. These
ζ -20 studies were performed under diffusion-controlled conditions at
pH 3.5 and 7.4. The number of latex particle adsorbed as a function
-40 of deposition time was determined by the direct counting proce-
dure using the optical microscopy and AFM.
A major advantage of the optical microscopy is that it enables
-60
measurements to be carried out under wet conditions, where par-
ticle positions over the substrate surface remain undisturbed. This
-80 allows one to reliably determine not only the coverage of particles
0.0 0.1 0.2 0.3 0.4 0.5
Θf but also their surface concentration and distributions over bare
and fibrinogen-covered substrates.
(b) 40 The statistical uniformity of colloid particle distributions was
confirmed quantitatively using variance analysis of the number
20 of particles deposited over equal-sized surface areas. This enabled
one to determine the coverage of latex particles on mica surfaces
precovered by fibrinogen sublayers and consequently the relation-
0
ship between the maximum (saturation) latex coverage HL and the
fibrinogen coverage Hf.
ζ -20 The procedure for determining HL was as described above. First,
fibrinogen monolayers of an appropriate density (determined by
-40 AFM) were produced on mica sheets under diffusion transport con-
ditions. Then, the wet sheets were immersed in the latex suspen-
-60 sion having a bulk concentration of typically 1.77  1010 cm3.
After completing the deposition run, typically lasting 24 h, the la-
tex distribution and coverage were independently determined by
-80
0.0 0.1 0.2 0.3 0.4 0.5 optical microscopy and AFM. It should be noted that in all these
Θf experiments, latex deposition was irreversible, as confirmed in
separate desorption runs, where the pure latex-covered mica
(c) 40 sheets were immersed for a prolonged time period (24 h) in pure
electrolyte having the same ionic strength and pH.
20 The dependencies of the maximum latex coverage HL on the
fibrinogen coverage Hf derived from these kinetic experiments
are plotted in Fig. 5 for pH 3.5 and in Fig. 6, for pH 7.4, respectively.
0
In order to compare results obtained for different ionic strengths,
the reduced maximum coverage HL/Hmx was used as the depen-
ζ -20 dent variable (where Hmx is the maximum coverage for a homoge-
neous surface) depending slightly on the ionic strength.
-40 As can be seen in Figs. 5 and 6, HL/Hmx was slightly dependent
on the ionic strength and increased monotonically with Hf . For pH
3.5 the increase of HL/Hmx with the fibrinogen coverage was quite
-60
abrupt, whereas for pH 7.4, the maximum latex coverage increased
at a much slower rate. Hence, an apparent threshold value of the
-80 Hf was needed to achieve significant latex deposition. For pH 7.4,
0.0 0.1 0.2 0.3 0.4 0.5
Θf this threshold value was approximately 0.04, whereas for pH 3.5,
it was significantly smaller, equal to 0.01. A similar behavior was
Fig. 4. The dependence of the zeta potential of mica f on the coverage of fibrinogen previously observed for latex particle adsorption on polyelectro-
Hf = SgN. The points denote experimental results obtained from the streaming lyte-covered mica [37,38].
potential measurements: (a) I = 103 M, pH 3.5, fi = 63 mV, ff = 28 mV; (b)
Analogous results, pertinent to the initial deposition rate of sil-
I = 102 M, pH 3.5, fi = 52 mV, ff = 24 mV. The solid lines represent exact theoretic
results calculated according to the electrokinetic model developed in Refs. [35,36]. ica particles on pDMAEMA and fibrinogen-covered silica slides,
(c) I = 102 M, pH 7.4. The solid lines represent exact theoretic results calculated were also reported [15]. In the latter case, the threshold value for
according to the electrokinetic model developed in Refs. [34,35] for fi = 80 mV, silica particle adsorption was roughly. 0.1 for I = 2.6  102 M
ff = 19 mV, and the dashed line for fi = 80 mV, ff = 30 mV. and 0.15 for I = 5  103 M. This difference stems from the fact that
our deposition experiments lasted 24 h, whereas the kinetic exper-
potential of adsorbed fibrinogen molecules is smaller (more nega- iments of Kalasin and Santore [15] were performed under convec-
tive) than in the bulk. Indeed, a good agreement between experi- tion conditions (in the parallel-plate channel) and lasted typically
mental data and the theoretic model is observed if the effective 5 min. These conditions were, therefore, less favorable for an effec-
zeta potential of fibrinogen equal to 30 mV is assumed (dashed tive attachment of silica particles under strong shearing forces pre-
line in Fig. 4c). As discussed in Ref. [22] this behavior can be ex- vailing at the channel’s walls.
plained in terms of a heterogeneous charge distribution over The results shown in Figs. 5 and 6 indicate unequivocally that
fibrinogen molecules. Thus, at pH 7.4, there apparently exists pos- latex particle immobilization was not possible at adsorption sites
460 Z. Adamczyk et al. / Journal of Colloid and Interface Science 356 (2011) 454–464

formed by only one fibrinogen molecule. If this were the case, HL/ 1.0
 1=2
2
Hmx should increase initially as k2 Hf =Hmx (where k ¼ pSag ), i.e.,
proportionally to Hf. Considering that in our case Sg = 1.28  0.8
1012 cm2 and pa2 = 5.03  109 cm2 one obtains k2 = 3.93  103.
Thus, the slope of the HL/Hmx vs. Hf dependence should be initially
very large initially and independent of pH. 0.6

ΘL/Θmx
It is interesting to note that the data shown in Figs. 5 and 6 are
anomalous in comparison to measurements of colloid particle
deposition on homogeneous surfaces [31,33]. This becomes evi- 0.4
dent if one notes that for pH 3.5 (see Fig. 6) and Hf < 0.15, where
a significant latex deposition was measured, the averaged zeta po-
0.2
tential of the fibrinogen-covered mica substrate remained negative
(see Fig. 4), i.e., of the same sign as the zeta potential of latex. This
should result in a strong electrostatic repulsion preventing particle 0.0
deposition. Qualitatively, this discrepancy could be explained in 0.0 0.1 0.2 0.3 0.4
terms of a heterogeneous distribution of positive charge due to Θf
natural fluctuations in adsorbed protein density. Consequently,
Fig. 6. Same as in Fig. 5 but for pH 7.4, I = 102 M. The points denote the
the local charge of the substrate could become positive, enabling
experimental results obtained by optical microscopy (d) and AFM (s).
latex particle deposition.
However, the results obtained for pH 7.4 (see Fig. 6) are even
more intriguing, because in this case all components involved in
the adsorption event, i.e., the bare mica substrate, fibrinogen mol-
is observed for negative values of the substrate surface zeta poten-
ecules, and latex particles exhibited negative zeta potentials.
tial, being of the same sign as the latex particle zeta potential.
Hence, paradoxically, the conjuncture of three negative interac-
It is interesting to compare this behavior with predictions
tions produced a positive effect, i.e., irreversible deposition of latex
stemming from the standard DLVO theory, which was previously
particles. This phenomenon can also be explained in terms of the
applied for successfully predicting particle deposition on homoge-
density fluctuation concept if a heterogeneous charge distribution
neous surfaces [31]. The main assumption of this theoretic ap-
over adsorbed fibrinogen molecules is assumed. Therefore, the
proach is that the van der Waals and electric double-layer
simple latex deposition experiments shown in Fig. 6 suggest quite
interactions are additive, which can be expressed for the spherical
unequivocally that at pH 7.4, where the averaged zeta potential of
particle/flat interface by the equation [34].
fibrinogen is negative (19 mV for I = 102 M), there were positive
patches on its surface. This supports the previous conclusion de- A123 a
/ðhÞ ¼  þ /0 eh=Le ; ð7Þ
rived from the streaming potential measurements [22]. 6h
The results shown in Figs. 5 and 6 can be interpreted in a more
where /ðhÞ is the particle / interface interaction energy, A123 is the
quantitative way if the normalized maximum coverage HL/Hmx is
Hamaker constant for the polystyrene/water/mica interactions, h is
plotted against the average zeta potential of fibrinogen-covered
the gap with between the particle and the interface, /0 ¼
mica f, rather than the fibrinogen coverage. The transformation
16aeðkT=eÞ2 tan hðfe=4kTÞ tan hðfp e=4kTÞ is the characteristic energy
from Hf to f was done using the results shown in Fig. 4.
for the linear superposition approximation (LSA) double-layer mod-
As seen in Fig. 7, a quite universal graph was obtained in this
el [39,40].
way, with experimental values of HL/Hmx fairly independent of
Assuming a typical value of A123 = 5  1014 kT, and f = 5 mV,
the ionic strength and pH. However, the results shown in Fig. 7
fp = 82 mV (see Table 1), I = 103 M one obtains from Eq. (7) that
are anomalous because a considerable deposition of latex particles
/ = 278 kT for h = 7 nm (fibrinogen diameter) and / = 0.1 kT for
h = 60 nm. Analogously, for I = 102 M, f = 5 mV, fp = 102 mV,
/ = 59 kT for h = 7 nm and / = 0.5kT for h = 15 nm. These estima-
tions indicate unequivocally that the energy barrier of considerable
1.0 height and extension of db = 15–60 nm is predicted to appear even
for such a low zeta potential of the substrate.
It is interesting to note that even for such low zeta potential of
0.8
the interface, the height of the barrier is mainly governed by the
electrostatic interactions.
ΘL/Θ mx

0.6 Knowing the specific energy distribution, one can calculate the
adsorption rate constant from the definite integral [33,34].
"Z #1
0.4 e/ðhÞ=kT
ka ¼ dh ; ð8Þ
db DðhÞ
0.2
where D(h) is the position-dependent diffusion coefficient of the la-
tex particles.
0.0 Knowing Ka, the latex coverage as a function of deposition time
0.00 0.05 0.10 0.15 0.20 0.25 0.30 and bulk concentration can be theoretically calculated by solving
Θf the governing diffusion equation using the finite-difference
scheme [33].
Fig. 5. The dependence of the reduced coverage of negative latex particles L800 HL/
Hmx on the fibrinogen coverage Hf. The points denote the experimental results
It is interesting to note that for a barrier height exceeding 10 kT
obtained by optical microscopy (d) and AFM (s) for ionic strength 103 M, (N, 4) (which is the case for f > 2 mV), the kinetics of latex particle
for ionic strength 102 M, pH 3.5, T = 293 K. deposition can be approximated by the expression.
Z. Adamczyk et al. / Journal of Colloid and Interface Science 356 (2011) 454–464 461

The basic assumption of the charge fluctuation approach is that


1.0 particle adsorption occurs at point-like sites distributed at random
over an otherwise homogeneous surface [41]. It is also assumed
that one site is capable of immobilizing one particle only. The basic
0.8
parameter of this approach, characterizing the density of adsorp-
tion sites (fibrinogen molecules in our case), is defined as
0.6 pa2
ΘL /Θmx

a ¼ pa2 Ns ¼ Hs ¼ k2 Hs ; ð10Þ
Sg
0.4
where Ns is the surface concentration of adsorption sites. As calcu-
lated above, in our case, k2 = 3.93  103.
0.2 Knowing the a parameter one can calculate the maximum cov-
erage of latex particles on a heterogeneous surface covered by sites
from the analytical dependence [23,41] modified in this work to
0.0 account for the fact that Hmx is a variable, rather than a constant.
-60 -40 -20 0 20 !
ζ 1 þ 0:314a2 þ 0:45a3
HL ¼ Hmx 1  : ð11Þ
Fig. 7. The dependence of the reduced coverage of negative latex particles L800,
1 þ H1mx a þ 0:66a3 þ a7=2
HL/Hmax on the zeta potential of fibrinogen-covered mica f determined by the
streaming potential method. The points denote the experimental results obtained In the limit of low coverage of sites, where a  1, Eq. (11) simplifies
by optical microscopy and AFM for: (d) I = 103 M, pH 3.5; (N) I = 102 M, pH 3.5; to
(4) I = 102 M, pH 7.4. The dashed line denotes theoretic results calculated from the
DLVO theory for a continuous zeta potential distribution. HL ¼ k2 Hs : ð12Þ
This approach was generalized in Refs. [37,38] assuming that an
 
D /b /b =kT effective particle immobilization can occur on sites formed by ns
HL ¼ Sg ka nb t ffi e Sg nb t: ð9Þ closely spaced molecules (polyelectrolytes or proteins). It was
a kT
shown in these works, assuming the Poisson statistics that the cov-
The results calculated using Eqs. (7)–(9), referred to as the DLVO erage of adsorption sites composed of ns or more particles is given
theory predictions, are shown by the dashed line in Fig. 7. As can be by the expression.
seen, for f < 0, the DLVO theory predicts negligible latex deposition,
after 24 h. This deviates completely from experimental results, X1
ðSHf Þn ðSHf Þns
Hs ðns Þ ¼ eSHf ffi ; ð13Þ
indicating a significant deposition for substrate zeta potential as n¼ns
n ns !
low as 50 mV.
It is interesting to note that such behavior was observed before where S ¼ S =Sg and S is the effective interaction area, whose size is
for heterogeneous surfaces produced by controlled cationic poly- comparable with the cross-sectional area of the site, thus S  1.
electrolyte adsorption [37,38]. As can be noted, the coverage of sites composed of two Fb mol-
As postulated in these works, the deviation from the DLVO the- ecules is proportional to H2f , three molecules H3f , etc. This means
ory is a direct manifestation of a discrete charge distribution that the number of such sites is very low for Fb coverage not
caused by natural fluctuations in the fibrinogen density over the exceeding 0.1.
mica substrate. Because fibrinogen molecules exhibited a net posi- Using Eqs. (13) and (11) one can predict that latex coverage in-
tive zeta potential at pH 3.5, a local increase in their concentration creases initially according to the relationship
at mica could lead to formation of favorable adsorption sites, capa-
Hnf s
ble of immobilizing latex particles. HL ¼ k2 : ð14Þ
Recently, deviations from the classical DLVO theory were theo- ns !
retically analyzed in Ref. [27] using the grid surface integration Thus, in the case of ns = 2, a parabolic dependence of H L on the
(GSI) technique. Both the surface roughness effect and the charge Fb coverage is initially predicted, described by the equation
heterogeity were quantitatively evaluated. The surface heterogene-
ities were assumed to have the form of nanopillars having opposite 1 1
HL ¼ kHs ð2Þ ¼ k2 H2f : ð15Þ
sign of zeta potential as the surface and colloid particles depositing 2 2
on it. A significant decrease in the energy barrier height with the In general, for arbitrary and ns, the latex coverage can be calcu-
size of the nanopillars was predicted for their coverage of 0.25. This lated from Eq. (11) using Eq. (13) to calculate Hs.
seems in a qualitative accordance with our experimental observa- As can be seen in Fig. 8, the experimental data are not properly
tions show in Fig. 7. However, the effect of the nanopillars coverage reflected by theoretic results derived from these equations if ns = 1
has not been studied in Ref. [27], which prohibits a quantitative is assumed, since a much steeper increase of HL/Hmx with the Fb
comparison with our experimental data. Moreover, these theoretic coverage was observed initially. This indicates that adsorption of
results cannot account for the situation arising for pH 7.4, where the latex particles on sites consisting of a single Fb molecule was not
net zeta potential of fibrinogen was negative. Since a significant possible due to too low attractive interaction energy. An analogous
deposition of latex was also observed in this case (see Fig. 6) one phenomenon was previously observed in the case of poly(allyla-
can suspect that there were positive patches on fibrinogen, which mine) hydrochloride and poly(ethylene imine) polyelectrolytes
were used to make permanent contacts with latex particles. [37,38], where latex particle deposition occurred at adsorption
These effects can be properly analyzed in terms of the charge centers composed of two or more closely spaced polyelectrolyte
fluctuation theory previously developed to interpret particle depo- chains.
sition on surfaces covered by polyelectrolytes [36,37] and fibrino- However, a quantitative agreement of theoretic results with the
gen molecules [23]. In a recent work [26], this approach was experimental data shown in Fig. 8 is attained when ns = 2 is as-
exploited to interpret the influence of ionic strength on silica par- sumed (see curve 2). In particular, the parabolic dependence, de-
ticle deposition rates on polyelectrolyte-covered silica surfaces. scribed by Eq. (15) for low Hf coverage, is confirmed. This
462 Z. Adamczyk et al. / Journal of Colloid and Interface Science 356 (2011) 454–464

suggests that the CE experiments of latex deposition on fibrinogen


1
monolayers can be exploited for an efficient determination of the 1.0
bulk concentration of the protein.
2
Analogous results obtained for pH 7.4 are shown in Fig. 9. As can
0.8
also be seen, the negative latex adsorbed efficiently for Hf > 0.04
(threshold value of fibrinogen coverage). As noted above, this can
be explained by assuming a heterogeneous charge distribution 0.6

ΘL/Θ mx
over Fb molecules, bearing patches of positive charge on their
surfaces.
Using the fluctuation theory, one can analyze this effect in more 0.4
detail by determining the number of Fb molecules necessary for
the immobilization of latex particles. As seen in Fig. 9, the experi- 0.2
mental data for pH 7.4 are in accordance with the theoretic predic-
tions derived from Eq. (13) assuming that adsorption sites were
formed by three Fb molecules (solid line in Fig. 10). It is interesting 0.0
to observe that for Hf < 0.1, the experimental data were well de- 0.00 0.05 0.10 0.15 0.20 0.25 0.30
Θf
scribed by the limiting analytical formula derived from Eq. (14)
for ns = 3, Fig. 8. The dependence of the reduced coverage of negative latex particles L800,
HL/Hmx on the fibrinogen coverage Hf. The points denote the averaged exper-
1
HL ¼ k2 H3f : ð16Þ imental results obtained by optical microscopy and AFM (s) for ionic strength
6 103 M, (N) for ionic strength 102 M, pH 3.5, T = 293 K. The solid lines 1–2 denote
theoretic prediction derived from the fluctuation theory, Eqs. (11) and (13) for the
Thus, in this case the latex coverage HL is expected to increase
adsorption site composed of one and two fibrinogen molecules, respectively. The
proportionally to the third power of the Fb coverage for the low dashed line denotes the limiting analytical solution, i.e., HL ¼ 12 k2 H2f =Hmx .
coverage range.
Immobilization of latex particles on sites composed of three Fb
molecules is understandable because at pH 7.4 (I = 102 M) the
partial positive charge on fibrinogen equals 12e [22] being consid-
1.0
erably smaller than the positive charge pH 3.5 (I = 102 M), esti-
mated to be 24e. However, one should remember that the partial 3

positive charge at pH 7.4 is overcompensated by the negative 0.8


charge, equal to 33e. Therefore, the experimental data shown in
Fig. 9 suggest strongly that positively charged patches over Fb mol-
ecules are separated by a considerable distance from negative 0.6
ΘL/Θmx

patches leading to a dipole formation. Considering the Fb molecule


structure and conformation [7–10] this could only happen if the
core region of Fb (D–E domains) was negatively charged, whereas 0.4
the side arms (aC domains) were positively charged. Hence, they
could act as anchors for the immobilization of Fb at the mica sur-
0.2
face. This concept is in accordance with the charge distribution
over Fb proposed in Ref. [9]. If such a charge distribution is true,
clusters of three Fb molecules forming a triangle could exhibit a 0.0
considerable positive charge (of about 36e) in the middle region, 0.0 0.1 0.2 0.3 0.4
which would be sufficient to immobilize irreversibly latex Θf
particles.
Such a heterogeneous charge distribution over Fb molecules can Fig. 9. Same as in Fig. 8, but for pH 7.4, 102 M. The solid line 3 denote theoretic
prediction derived from the fluctuation theory, Eqs. (11) and (13) for the adsorption
also explain why the standard DLVO theory does not work in the site composed of three fibrinogen molecules. The dashed line denotes the limiting
case of the negative latex deposition for pH 7.4, where the average analytical solution, i.e., HL ¼ 16 k2 H3f =Hmx .
zeta potential of the mica substrate remains negative for the entire
range of Fb coverage.
It is also interesting to observe that the results in Fig. 9, showing high coverage range, fluctuations in the Fb molecule density (local
a more gradual increase of latex coverage with Hf , can be exploited charge) within the area of interaction with the latex particles
for a sensitive determination of the bulk Fb concentration by the (whose cross-sectional area is k2 = 3.8  103 times larger than the
colloid deposition method. Knowing HL, this can be done using fibrinogen molecule cross-sectional area) become negligible. As a
Eq. (11) to calculate the Fb coverage Hf and then Eq. (3) to calcu- result, the local zeta potential of Fb-covered mica remained close
late cb for a fixed fibrinogen adsorption time. to the averaged zeta potential.
In order to explore further the potential of the colloid deposi- The strict correlation of positive latex deposition with the zeta
tion method for studying Fb layers, an additional series of deposi- potential of fibrinogen-covered mica demonstrated in Fig. 10 can
tion experiments was performed using the positive latex A800. The be exploited for a precise determination of the isoelectric point
results of these measurements are presented in Fig. 10 for pH 3.5 of protein-covered surfaces, which is of essential practical
and in Fig. 11 for pH 7.4. As can be seen, for pH 3.5 and Hf < 0.16 significance.
the positive latex coverage remained practically the same as the The results shown in Fig. 10 also prove that the negative and po-
limiting value for homogeneous surfaces. For Hf > 0.16, the latex sitive latex deposition on Fb layers is a complementary process be-
coverage decreased abruptly to negligible values. Hence, in this cause for Hf < 0.16 the positive latex adsorbs fully (making a
case, the latex deposition corresponded with the average zeta po- negative replica of the Fb monolayer) and for Hf > 0.16 the nega-
tential of the mica substrate. Therefore, the DLVO theory proved tive latex adsorbs fully, making a positive replica. This behavior
adequate (see dashed line in Fig. 10). This was so, because for the can be utilized for performing a qualitative but very sensitive
Z. Adamczyk et al. / Journal of Colloid and Interface Science 356 (2011) 454–464 463

1 1
1.0 1.0
2

0.8 2 0.8

0.6 0.6
ΘL/Θ mx

ΘL/Θmx
0.4 0.4

0.2 0.2

0.0 0.0
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.0 0.1 0.2 0.3 0.4
Θf Θf

Fig. 10. The dependence of the reduced coverage of latex particles HL/Hmx on the Fig. 11. Same as in Fig. 10 but for pH 7.4, 102 M. The points denote the averaged
fibrinogen coverage Hf for pH 3.5, T = 293 K. The points denote the averaged experimental results obtained by optical microscopy and AFM for (d) the L800
experimental results obtained by optical microscopy and AFM for (d) the L800 latex, and (O) the A800 latex. The solid line 2 denotes theoretic predictions derived
latex, I = 103 M and (N) 102 M; (}) the A800 latex, I = 103 M and (O) 102 M. The from the fluctuation theory, Eqs. (11) and (13), and the dashed line 1 denotes the
solid line 2 denotes theoretic predictions derived from the fluctuation theory, Eqs. results predicted from the DLVO theory for the A800 latex.
(11) and (13), and the dashed line 1 denotes the results predicted from the DLVO
theory for the A800 latex.

It was also concluded that the colloid deposition method can be


used for a quantitative determination of the Fb bulk concentration,
detection test of fibrinogen layers on mica, and, therefore fibrino-
for a range inaccessible for other methods.
gen presence in the bulk.
Another practical significance which can be drawn from these
results is that for fibrinogen coverage range of 0.02 < Hf < 0.16, Acknowledgments
both the negative and the positive latexes will deposit with a
comparable efficiency. Hence, such surfaces can be named super- This work was supported by the COST Action D43 Special Grant
adsorbing surfaces, because they are able to remove both posi- and Grant No. POIG.01.01.02-12-028/09-01. The authors are
tive and negatively charged colloid particles from their indebted to Dr. E. Bilańska for performing SEM measurements of
suspensions. This could be of vital significance for various filtra- latex monolayers.
tion processes.
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