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Biomolecular Engineering 24 (2007) 119–124

Early stages of human plasma proteins adsorption probed

by Atomic Force Microscope

K. Mitsakakis, S. Lousinian, S. Logothetidis *

Aristotle University of Thessaloniki, Department of Physics, Solid State Physics Section, GR-54124 Thessaloniki, Greece

Abstract

Atomic Force Microscope (AFM) as a surface characterization technique has offered a great impulse in the advance of biocompatible materials.

In this study AFM was implemented for the investigation of the early stages of adsorption of two human plasma proteins on titanium and

hydrogenated carbon biocompatible thin films. The plasma proteins that were used were Human Serum Albumin and Fibrinogen, two of the most

important proteins in human plasma. The concentration of the protein solutions was the same as that in human plasma. As the examined samples

were soft, non-contact AFM mode was used to avoid their destruction. In order for the early stages of protein adsorption to be assessed, small

incubation times were applied. AFM measurements in liquid buffer were also carried out, allowing the observation of the protein behaviour in an

environment much closer to their native one. In addition, there was an assessment of the adsorption mechanism of the proteins on the above-

mentioned biomaterials.

# 2006 Elsevier B.V. All rights reserved.

Keywords: Plasma proteins; Atomic Force Microscopy; Human Serum Albumin; Fibrinogen

1. Introduction

Atomic Force Microscopy (AFM) (Binnig et al., 1986) has

been implemented as a surface characterization technique for

the examination of biomolecules in various cases in biology-

biomaterial research (Hansma et al., 1997; Frederix et al., 2004;

Vansteenkiste et al., 1998). In the present work, it was involved

in the investigation of human plasma protein adsorption on

various biocompatible thin films. AFM offers the significant

advantage of probing in high detail the surface topography

qualitatively (by surface images) and quantitatively (by

mathematical quantities like surface roughness) due to its

nanometer-scale spatial resolution, both lateral and vertical

(Erlandsson et al., 1988; Hansma et al., 1988; Jandt, 2001).

AFM has proved to be very helpful for the determination and

verification of various morphological features and parameters,

like special molecular shapes and protein clusters, cluster size,

surface coverage, etc.

Protein adsorption, on the other hand, is a process that takes

place in effect on the interface of liquid solution and solid

substrate. In general, it is a quite multi-parametric procedure as

it is regulated by numerous factors like hydrophobicity, pH,

* Corresponding author. Tel.: +30 2310 998174; fax: +30 2310 998390.

E-mail address: logot@auth.gr (S. Logothetidis).

1389-0344/$ – see front matter # 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.bioeng.2006.05.013

surface roughness, chemical composition, etc., and this

diversity of parameters makes it complex (Silva, 2002; You

and Lowe, 1996). However, it is of great importance, as a

process, when it comes to contact of biological matter, like

human plasma proteins which is of interest in this study, with a

biomaterial. Especially in haemocompatibility studies, the

adsorption of plasma proteins plays a key role, as it is the

proteins themselves that first come into contact with the

external biomaterial and promote or prevent the formation of

thrombus at the site of the ‘‘foreign’’, to plasma, biomaterial.

Human Serum Albumin (HSA) and Fibrinogen (Fib) are two of

the most important human plasma proteins. More particularly,

Fib takes part in blood coagulation, facilitates adhesion, and

aggregation of platelets, and is important in the processes of

both haemostasis and thrombosis, whereas HSA is believed to

act controversially to Fib, although its specific action is not yet

clear (Cacciafesta et al., 2000; Ortega-Vinuesa et al., 1998).

That is the reason why these two proteins were selected for the

purpose of our study.

Experiments have shown that amorphous hydrogenated

carbon (a-C:H) exhibits haemocompatible behaviour, and that

is why the main study was focused on a-C:H; Ti was

implemented as a reference material. Therefore, we were

motivated to use these materials as substrates for protein

adsorption (Logothetidis et al., 2005; YuU et al., 2000;

Vinnichenko et al., 2004; Logothetidis, 2002).

K. Mitsakakis et al. / Biomolecular Engineering 24 (2007) 119–124120

2. Experimental

Titanium and a-C:H thin films were used for the study of protein adsorption.

More particularly, there were two types of a-C:H thin films, both deposited on c-

Si(1 0 0) wafer with RF reactive magnetron sputtering in a high vacuum

chamber with H2 (10%) as the reactive gas; one was deposited with biased

voltage (�40 V) and the other was deposited with no substrate bias (floating)

(Logothetidis, 2002; Gioti and Logothetidis, 2003).

The two proteins that were used in the experiments are HSA and Fib. HSA is

a heart-shape protein with approximate dimensions of 8 nm � 8 nm � 3 nm

(Sugio et al., 1999), while Fib is a more extended molecule with

length � width � height approximately 50 nm � 6 nm � 9 nm (Jandt, 2001;

Cacciafesta et al., 2000). Solutions of HSA and Fib in phosphate-buffered saline

(PBS, pH 7.4) were prepared and the incubation procedure was as follows; a-

C:H thin films were dipped into the protein solutions then the samples were

rinsed with deionized water and dried with mild N2 flow. The concentrations of

the solutions were 40 and 5 mg/ml for HSA and Fib, respectively—these are the

concentrations in human plasma as well. For both different samples, the

solutions were left for 5 and 10 min. Complementary measurements with

AFM were performed in liquid buffer as well.

Measurements were performed with SOLVER P47H Scanning Probe Micro-

scope (NT-MDT, NTI Instruments) in ambient and liquid environment. For

measurements in air, standard silicon cantilevers with nominal spring constant

11 N/m and resonance frequency 227 kHz were used for both cases. Because

protein samples are delicate and easy to be destroyed by the sharp AFM tip, Semi/

Non-Contact operation mode was employed. This way, the oscillatory motion of

the tip above the surface allows practically no contact with the sample and

therefore no danger to drag, deform, or scratch the latter exists. When it comes to

liquid measurements, contact operation mode was utilized. Cantilevers with

spring constant as soft as 0.1 N/m probed the surface morphology of the proteins

in their near-physiological environment. In either case, square images of the

samples were taken, with various sizes; 1 mm � 1 mm and 500 nm� 500 nm or

less when they would reveal interesting information. Quantities that were used for

the evaluation and comparison of the acquired data are peak to valley distance

(peak-to-peak), and root-mean-square roughness (Rrms).

3. Results and discussion

The haemocompatibility study of the a-C:H thin films used

in this work (through Spectroscopic Ellipsometry) has shown

that the film deposited under floating conditions presents much

Table 1

The film types, incubation times, and morphology parameters of surfaces,

including peak-to-peak distance and RMS roughness

Film type Incubation

time (min)

Peak-to-peak

(nm)

RMS roughness,

Rrms (nm)

a-C:H (biased) 0 5.160 0.566

HSA/a-C:H (biased) 5 13.980 1.255

HSA/a-C:H (biased) 10 6.695 0.842

Fib/a-C:H (biased) 5 19.020 2.363

Fib/a-C:H (biased) 10 6.630 0.910

a-C:H (floating) 0 15.840 2.131

HSA/a-C:H (floating) 5 24.900 2.758

HSA/a-C:H (floating) 10 16.200 2.365

Fib/a-C:H (floating) 5 21.840 2.776

Fib/a-C:H (floating) 10 20.526 2.576

Ti 0 10.560 0.994

HSA/Ti 5 13.140 1.022

HSA/Ti 10 10.560 1.048

Fib/Ti 5 16.260 1.849

Fib/Ti 10 12.000 1.278

HSA/a-C:H (floating) In liquid 14.040 1.846

Fib/a-C:H (floating) In liquid 17.940 2.286

better haemocompatible behaviour than the one deposited

under application of negative bias voltage (Logothetidis et al.,

2005). There has also been a study of protein adsorption

through AFM technique for longer incubation times (>10 min)

(Lousinian et al., 2007). This is the reason why the early stages

of protein adsorption is studied on these two samples, in this

work. The parameters that varied were: (1) different thin film

for protein adsorption; (2) different protein that was studied; (3)

different incubation times. Therefore, comparisons of results

will be made on three directions, each time keeping the rest of

two constant. In Table 1 and Fig. 1 details about peak to valley

distance and RMS roughness are presented, for incubation

times 0 min (bare substrate), 5, and 10 min, for HSA and Fib

proteins. This is why there will be several references to them

within this work.

From Table 1 and Fig. 1 it is noticed that the range of Rrms

values in the case of biased a-C:H is wider than in the case of

floating a-C:H. The relative changes in Rrms for Fib and HSA

from 0 to 5 min incubation times are 317.5 and 121.7%,

respectively, for the case of biased a-C:H. Equivalently, the

relative changes in Rrms for Fib and HSA from 0 to 5 min

incubation times in the case of floating a-C:H are only 30.3 and

29.4%, respectively. The large difference in relative roughness

values between biased and floating a-C:H thin films (for 5 min

incubation time) does not necessarily imply that there is more

protein material adsorbed on the former than the latter.

However, the fact that the surface roughness increases in both

cases, does mean that during the first 5 min there is definitely

some amount of proteins adsorbed on the surfaces.

By measuring and comparing the ‘‘bare’’ a-C:H substrates

through AFM images (Figs. 2a and 4a), it is clearly observed

that floating a-C:H substrate exhibits higher surface roughness

than the biased a-C:H, which is expected, since the sputtered

carbon atoms are distributed more evenly on Si substrate under

the influence of bias voltage.

Fig. 1. Comparative diagram of RMS roughness of examined plasma proteins

vs. incubation time for the two different types of a-C:H thin films and Ti thin

film.

K. Mitsakakis et al. / Biomolecular Engineering 24 (2007) 119–124 121

Fig. 3. Topography image of: (a) Fib on a-C:H (biased) after 5 min incubation time (scan size 1 mm � 1 mm). Arrows indicate the molecular cluster features of

fibrinogen, (b) Fib on a-C:H (biased) after 5 min incubation time, with a 500 nm � 500 nm size focus on one of the fibrinogen molecular cluster—shape features

(Inset: the 2D equivalent image), and (c) Fib on a-C:H (biased) after 10 min incubation time (scan size 1 mm � 1 mm).

Fig. 2. 1 mm � 1 mm topography image of: (a) a-C:H (biased) as deposited, (b) HSA on a-C:H (biased) after 5 min incubation time (Inset: 500 nm � 500 nm size focus

on protein cluster), and (c) HSA on a-C:H (biased) after 10 min incubation time (Inset: 3D topography image, where the height differences are more clearly visible).

Fig. 2a shows the surface of biased a-C:H film without

proteins. It appears to have grain-like surface features, the size of

which is around 20–30 nm. Fig. 2b is the 5-min image of HSA on

biased a-C:H, and it is seen clearly that protein aggregates exist.

Typical dimensions are 100 nm � 180 nm and 11 nm height for

the bigger aggregate and 70 nm � 70 nm and 8 nm in height for

the small one, whereas the yellow circles denote regions with

other aggregates, with dimensions approximately 70–80 nm and

7–8 nm in height (it is worth noticing that in all the above-

mentioned cases, the proteins tend to ‘‘spread laterally’’, rather

than forming hills by accumulation of one on the other, since the

Fig. 4. 1 mm � 1 mm topography image of: (a) a-C:H (floating) as deposited, (b)

(floating) after 10 min incubation time.

height of the clusters is approximately once or twice the height of

one HSA molecule, while the lateral dimensions are larger).

Fig. 2c shows that in 10 min of incubation time, there are larger

protein clusters and the surface is partially covered. This is also

quantitatively verified by the decrease (even small) in the Rrms of

HSA, since aggregates dispersed on a relatively smooth surface

increase its roughness.

It was mentioned before that the Rrms of biased a-C:H thin

film is lower than that of floating a-C:H. That allows the

observation of molecular features of adsorbed proteins on

biased a-C:H. More specifically, in Fig. 3a the AFM reveals, in

HSA on a-C:H (floating) after 5 min incubation time, and (c) HSA on a-C:H

K. Mitsakakis et al. / Biomolecular Engineering 24 (2007) 119–124122

Fig. 5. 1 mm � 1 mm topography image of: (a) Fib on a-C:H (floating) after 5 min incubation time and (b) Fib on a-C:H (floating) after 10 min incubation time.

fact, the packing of fibrinogen molecules. Because such a detail

is of great interest, images at smaller scan sizes (Fig. 3c) were

taken in order to focus on those regions. It was revealed, thus,

that the three-lobe structures that appear, are indeed, small

cluster of few molecules of fibrinogen – forming either

extended or V-shape conformations – that still retain the shape

of a single molecule (the size and morphology features fit those

mentioned in bibliography on TiO2 or mica (Jandt, 2001;

Cacciafesta et al., 2000; Marchant et al., 2002)). Such

‘‘protrusions’’ of the molecules cause the surface roughness

to increase, whereas in the case of 10 min incubation time

(Fig. 3b), Rrms is lower, due to the fact that more protein

material is deposited on biased a-C:H, ‘‘smoothening’’, thus, its

morphology (Fig. 1).

As far as the floating a-C:H thin film is concerned, the grain

sizes have maximum size, again, around 30–40 nm (Fig. 4a) (in

contrast with maximum 20–30 nm in biased a-C:H films) and

the percentage of big grains is greater than in the case of biased

a-C:H. These differences account for the 3.5� greater Rrms or

floating a-C:H thin films.

Comparing now the images of 5 and 10 min of incubation time

of HSA on floating a-C:H (Fig. 4b and c) one can note that both

the number and the size of the protein clusters are greater, which

means that more surface coverage is achieved. It cannot be

securely deduced, however, what percentage of surface has been

Fig. 6. 1 mm � 1 mm topography image of (a) Ti (as deposited), (b) HSA on Ti af

cluster), and (c) Fib on Ti after 5 min. Incubation time.

covered; only that in 10 min floating a-C:H is more covered,

relatively to the incubation time of 5 min. Decrease of Rrms

indicates gradual completion of surface coverage in these early

stages of protein adsorption (which may not be total till the

10 min, but certainly is more than in 5 min) during which both

protein–protein and surface–protein interactions are important.

From quantitative results from images of fibrinogen

solution of 5 min incubation on floating a-C:H (Fig. 5a), it

comes that Rrms for Fib- and for HSA-covered surface (both for

5 min and floating a-C:H film) is around the same value,

whereas the former is twice higher than the latter (2.36 nm

versus 1.26 nm) for the case of biased a-C:H film under the

same incubation conditions (Fig. 1). In addition, it is

remarkable that no special features are seen, as in the

equivalent case of biased a-C:H. One explanation is that the

surface roughness of the film is so high that does not allow any

shaped features to be ‘‘visible’’. On the other hand, another

possible case is that fibrinogen does not adsorb on floating a-

C:H in the same way that it does on biased a-C:H. However, this

speculation needs further investigation.

Although the main material of investigation was a-C:H, Ti

films on Si(1 0 0) were used as well as a substrate for protein

adsorption. From Table 1 it can be seen that Rrms values

for HSA on Ti are practically the same for both incubation

times. This ‘‘unchanging’’ behaviour could mean that since,

ter 5 min. incubation time (Inset: 200 nm � 200 nm focus on the HSA protein

K. Mitsakakis et al. / Biomolecular Engineering 24 (2007) 119–124 123

Fig. 7. 1 mm � 1 mm topography image in PBS liquid buffer of: (a) HSA on a-C:H (floating) in and (b) Fib on a-C:H (floating).

by the addition of a protein solution on the surface of Ti, hardly

any changes on its morphology take place, HSA adsorption on Ti

films is not favoured. On the contrary, Rrms for fibrinogen is

nearly doubled, revealing that changes on the surface of Ti took

place, i.e., Fib adsorption. After all, from Fig. 6b and c it can be

seen that in 5 min, only few HSA clusters exist (raising, thus,

negligibly the Ti substrate roughness), whereas for the same

time elapsed, several Fib aggregates – with size from 40 nm

and more, in contrast to the relatively small Ti grains of 30 nm

maximum size – are dispersed on Ti surface.

Finally, for qualitative reasons, measurements in liquid

were conducted. The processes of sample-to-tip approaching,

finding an appropriate region and scanning took several

minutes after the incubation of the solution drop on

the floating a-C:H surface, and due to that inherent limitation

in time that was imposed, the results are not indicative of

the early stage adsorption procedure (images were succeeded

to obtain in 30–35 min after incubation). Yet, they give a

qualitative view of the biomaterial surface when proteins are

in their near-physiological environment. What Fig. 7a and b

show, in fact, is that after several minutes of incubation time,

the protein–protein interactions prevail, since protein atoms

of the solution ‘‘meet’’ protein atoms on the surface as

they adsorb (instead of biomaterial atoms) and, thus, due to

the affinity of a protein molecule with the rest of its kind, the

surfaces show nearly the same morphology. However, Rrms

for Fib is somewhat greater than that of HSA, possibly due to

uneven settling of fibrinogen molecules on one another

and due to the fact that fibrinogen molecules are quite larger

than HSA.

4. Conclusions

From the present study, it is verified that protein adsorption

of HSA and fibrinogen is substrate dependent. This fact is

apparent both qualitatively (from images, by noting differences

in surface coverage, protein cluster sizes, protein morphology

features, etc.) and quantitatively (from variations in RMS

roughness). Fig. 1 indicates that Rrms exhibits an increase and

decrease at 5 and 10 min of incubation time, respectively,

nearly for all the examined cases. In addition, the Rrms values

for 10 min resemble those of bare floating a-C:H. These could

lead to the conclusion that at first, protein clusters form on

biomaterial surface, at distance from one an other, and then, as

protein material is added, they coalesce to form (at greater

incubation times possibly) a protein layer that fully covers the

surface, although the fact that both a-C:H and Ti have a grain-

like surface morphology (unlike mica or HOPG, which are

atomically smooth) makes it difficult to evaluate precisely the

degree of surface coverage by proteins.

It is obvious that the questions on protein adsorption cannot

be answered by a single technique, and require real-time and

kinetic experiments as well, but with this work it was proved

that AFM can contribute significantly to this direction. Further

steps on this work include verification of the above results with

contact angle measurements as well as imaging with Scanning

Near-Field Optical Microscopy. The former will assist the real-

time evaluation of the adsorption rate and the observation of the

profile of a protein solution drop, while the latter will exhibit

the competitive adsorption behaviour in the case incubation in

solution with both proteins.

Acknowledgement

One of us (K.M.) acknowledges financial support of Public

Benefit Foundation Alexander S. Onassis.

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