Biosensors and Bioelectronics 21 (2005) 7986

Quartz crystal microbalance-with dissipation monitoring (QCM-D) for

real time measurements of blood coagulation density and immune
complement activation on articial surfaces
Marcus Andersson
a,
, Jonas Andersson
b
, Anders Sellborn
a
, Mattias Berglin
a
,
Bo Nilsson
b
, Hans Elwing
a
a
Department of Cell and Molecular Biology/Interface Biophysics Lundberg Laboratory, G oteborg University, Box 462, SE-405 30 G oteborg, Sweden
b
Department of Oncology, Radiology and Clinical Immunology, Section of Clinical Immunology, Rudbeck Laboratory C5,
Uppsala University Hospital, SE-751 85 Uppsala, Sweden
Received 24 June 2004; received in revised form 24 September 2004; accepted 29 September 2004
Available online 11 November 2004
Abstract
Arecently developed variant of quartz crystal microbalance (QCM) called QCM-with dissipation monitoring (QCM-D) allows simultaneous
and simple measurements of changes in adsorbed mass as well as the viscoelastic property (D-factor) of deposited protein layers on the sensor
surface. We have taken the QCM-Dtechnology a step further and demonstrated its advantages in the study of protein assembly as a consequence
of surface induced immune complement activation, or contact activated blood coagulation. In the present study we have continued our QCM-
D investigations of surface assembly of brin clot formation and complement activation and incubated differently modied quartz sensor
surfaces in blood plasma and sera. Polymer surfaces used were spin-coated polyethylene, poly(ethylene terephtalate), poly(methylmetacrylate)
and poly(dimethylsiloxane). Also used were sputtered titanium and heparin grafted surfaces. In this investigation we found that we could
describe the surface induced coagulation with four independent parameters: (1) Time of onset of coagulation, (2) brin deposition rate, (3)
total frequency shift at stable plateau, and (4) brin clot density. The most important nding was that the blood plasma clot density can be
assessed with the use of D determinations and that the clot density varied signicantly with the chemical composition of the surface. However,
the D-factor did not give any new analytical information about the possible complement activation mechanisms. Nevertheless, the QCM-D
was found to be a reliable tool for the analysis of surface induced complement activation.
We also compared the QCM-D technique with traditional enzyme immuno assay (EIA) measurements of soluble products from the surface
activation of the complement and coagulation systems. We found that the results from EIA and QCM-D measurements corresponded well for
the complement activation but not for the coagulation, probably due to the biological complexity of the coagulation system.
2004 Elsevier B.V. All rights reserved.
Keywords: Quartz crystal microbalance; Blood coagulation; Complement activation; Enzyme immuno assay
1. Introduction
Quartz crystal microbalance (QCM) is a high frequency
surface sensitive method for various biosensor applications
(Marx, 2003). Most applications monitor only changes of
resonant frequency (f) of the adsorbed layers on the sensor

Corresponding author. Tel.: +46 31 7732566; fax: +46 31 7732599.

E-mail address: marcus.andersson@gmm.gu.se (M. Andersson).
surfaces. Recently, a variation of QCM has been developed
which simultaneously allows registration of dissipation (D)
of the sensor signal (Rodahl et al., 1995; Hook et al., 1998).
The D-factor is related to the viscoelastic properties of the ad-
sorbed layer. The new QCM modication, called QCM with
dissipation monitoring or QCM-D, has greatly increased the
analytical range of the instrument in different applications,
for example in studies of single protein adsorption (Hook et
al., 1998) and adsorption of phospholipid layers (Glasmastar
0956-5663/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bios.2004.09.026
80 M. Andersson et al. / Biosensors and Bioelectronics 21 (2005) 7986
et al., 2002). The benet of simultaneous registration of D
and f has been demonstrated in several investigations, where
QCM-D measurements were combined with optical surface
sensitive methods such as ellipsometry and surface plasmon
resonance (SPR) (Hook et al., 2001).
Our present interest in QCM-Dis mainly focused on mea-
surements of protein adsorption to biomaterials. Previously,
our group has used QCM-Dto study complex proteinsurface
interactions such as immune complement activation and
contact activated blood coagulation (Sellborn et al., 2003;
Andersson et al., 2002; Berglin et al., 2004). Surface induced
activation of the complement system and coagulation system
result in a massive deposition of proteins on the surface,
which is of great importance for biomaterial compatibility
(Tang et al., 1998; Kalltorp et al., 1999). Traditional methods
investigating complement and blood coagulation systems
are mostly based on measurements of soluble products.
The introduction of the QCM-D method presents a way of
studying new mechanisms of bio-interface research.
Aim of this investigation was to rene the QCM-D
technique for quantication of both the complement and the
coagulation system on solid surfaces. Preliminary studies
indicated that the clot density is dependent on the chemistry
of the surface. Thus, our second aim was to perform a more
detailed analysis of the D-parameter in relation to brin
deposition. The QCM-D was used to investigate materials
commonly used in medical implants such as titanium (Ti),
polyethylene (PE), poly(methyl methacrylate) (PMMA),
poly(ethylene terephtalate) (PET), poly(dimethylsiloxane)
(PDMS) and heparinized surfaces (Hep). The QCM-D
results were compared to results obtained from traditional
methods such as enzyme immuno assays (EIA) of soluble
factors and platelet counting.
1.1. Short description of the coagulation and
complement systems in relation to biomaterials
Activation of the blood coagulation system on biomate-
rial surfaces may result in brin clots that may impair the
function of various medical devices such as articial blood
vessels, stents (Christensen et al., 2001), blood catheters and
other extra corporal devices. Blood coagulation kinetics and
clot characterisation are studied in various ways such as spec-
trophotometry (Sanchez et al., 2002), QCM(Andersson et al.,
2002), ellipsometry (Walivaara et al., 1996), SPR (Hansson
et al., 2002; Vikinge et al., 2000a, 2000b) and free oscilla-
tion rheometry (Hansson et al., 2002). Platelets are important
mediators in the coagulation cascade and are involved in sur-
face induced blood coagulation. The platelet number is usu-
ally determined by cell-counting methods, and their degree
of activity is measured by the amount of thrombospondin
(TSP) released, using EIA or equivalent methods (Bergseth
et al., 2000). In our setup, EIAis performed on blood that has
been incubated on coated glass slides using the slide chamber
model (Hong et al., 1999b, 2001). Measuring potential sur-
face induced blood coagulation often involves measurement
of the soluble thrombinantithrombin complex (TAT) with
EIA as the standard method. However, the QCM-D instru-
ment has turned out to be a very interesting alternative for
determination of surface induced blood coagulation, since
the brin formation can be detected in situ and in real time
on the inductive surface. The frequency shift gives informa-
tion about the general clotting kinetics, such as time of onset
and brin deposition rate, while the dissipation factor pro-
vides valuable data concerning viscoelastic properties, such
as clot density. This way of measuring surface induced blood
coagulation has been further rened in this study.
The immune complement system is present in blood and
serum and consists of about 20 different proteins. On contact
with a foreign surface, e.g. a bacterial surface, the comple-
ment system is activated in a cascade fashion, resulting in
destruction of the bacterial surface or release of bioactive
degradation products, causing inammatory reactions in the
surrounding tissue, or both. However, the activated comple-
ment cascade can be a major problem concerning medical
implants and extra corporal blood treatment devices such as
oxygenators, dialysators, etc. (Tang et al., 1998; Kalltorp et
al., 1999). It also seems to have long term implications, e.g.
for in wear debris from total joint arthroplasty (DeHeer et
al., 2001) and xenotransplantation (Bengtsson et al., 1998).
Determination of complement activity usually involves EIA
measurements of fragments fromdegradation of complement
factors in blood or serum that has been in contact with an ac-
tivating surface. Examples of such products are complement
factor 3a and 5a (C3a and C5a) and the terminal comple-
ment complex (TCC). Complement activation on a solid sur-
face can also be detected by the amount of surface bound
complement factor 3 (C3) on the activating surface. Surface
associated C3 can be measured via anti-C3 antibodies, us-
ing a method such as ellipsometry (Elwing et al., 1986),
surface plasmon resonance (SPR) or QCM (Sellborn et al.,
2003).
2. Materials and methods
2.1. Surfaces
The following surfaces were used: titanium (sputtered
on gold sensor surfaces in vacuum), polyethylene (Aldrich,
WI, USA, 9002-88-4), poly(ethylene terephtalate) (Aldrich,
29154-49-2) poly(methyl methacrylate) (Aldrich, 9011-14-
7), and poly(dimethylsiloxane) (Rhodia silicones, France,
CAF 2534). Solvents and polymer concentrations are listed
in Table 1. The polymers were spin-coated (spin-coater KW-
4A Chemat Technology Inc., Northridge, CA, USA) on gold
sensor surfaces (Q-sense, G oteborg, Sweden) using 50 l
polymer solution (polyethylene was heated to approximately
200

C before casting). The thickness was estimated using

QCM-Din air (Table 1). Titaniumplates and microscope pur-
pose glass slides coated with the above-mentioned polymers
were used in the slide chamber model (see below). Surfaces
M. Andersson et al. / Biosensors and Bioelectronics 21 (2005) 7986 81
Table 1
General polymer characteristics; mean values of advancing and receding contact angle measurement as measured by the Wilhelmy plate method (n =2)
Polyethylene
(PE)
Poly(ethylene
terephtalate) (PET)
Poly(methyl
methacrylate) (PMMA)
Poly(dimethylsiloxane)
(PDMS)
Advancing contact angle (

) 103 70 75 103
Receding contact angle (

) 50 48 59 91
Glass transition temperature, T
g
(C

) 125 81 95 123
Melting transition temperature, T
m
(C

) 138 252 Amorphous 60

QCM sensor surface spin-coating premises 1% in decaline 1% in phenol 4% in toluene 1% in toluene
3000 rpm 5000 rpm 2000 rpm 2000 rpm
2 min 2 min 2 min 20 s
Estimated polymer thickness 2030 nm 20 nm 15 nm 84 nm
Glass transition temperature (T
g
) and melting transition temperature (T
m
) values were provided by the manufacturer. Polymer thickness was estimated by
QCM-D measurements in air.
were used only once, except the titaniumsurfaces which were
reused after they were thoroughly washed with sodiumdode-
cylsulfate (2%, 30 min), then excessively rinsed with MilliQ
water and placed in an UV-ozone oven (10 min) and nally
rinsed again in MilliQ water.
Heparinized surfaces (Corline Systems AB, Uppsala,
Sweden) were used as a non-complement activating sur-
face and served as negative control for all experiments
(Christensen et al., 2001). The heparinized surfaces were
prepared by irreversible adsorption of a macromolecular
conjugate that according to the manufacturer contains 70
heparin molecules covalently linked to a 50 kDa polymer
carrier (polymeric amide) (data on le, Corline Systems
AB, Uppsala, Sweden). The conjugate was then adsorbed by
electrostatic coupling to a polymeric amide coated surface.
The surface concentration of heparin was approximately 0,
5 g/cm
2
, which is equivalent to a lm thickness of about
10 nm. The antithrombin (AT) binding capacity reaches
about 24 pmol AT/cm
2
.
2.2. Dynamic and static contact angle measurements
Advancing and receding contact angle was determined
with the Wilhelmy plate method (Garbassi et al., 1994) using
de-ionized water ( =72.8 mJ/m
2
) at 20

C. The instrument
was a DCA-322 from Cahn operating at 100 m/s. Data was
evaluated with WinDCA Version 1.03 (Cahn, WI, USA). All
surfaces were tested with regard to hydrophobicity by the
sessile water drop technique (Dahlgren and Sundqvist, 1981)
to ensure complete coating coverage and to ensure clean ti-
tanium surfaces.
2.3. Blood products
Citrated blood plasma from an apparently healthy donor
(tested by the blood central, Sahlgrens University Hospital,
G oteborg, Sweden) was distributed to Eppendorf vials and
frozen (70

C). Serum was prepared from plasma mixed

with CaCl
2
(to a nal concentration of 25 mM) and incubated
for 30 min in 37

C. This procedure was carried out in order

to remove the brin from the plasma. The un-clotted prod-
uct was centrifuged (Universal 16R, 5200 rpm, 3000 g) for
30 min in 37

C. The pellet was discarded and the serum

was collected in Eppendorf vials and immediately frozen
(70

C) until further use. Thawed plasma and serum was

used immediately.
Blood used in the slide chamber model was drawn in the
lab (50 ml) from the same donor as mentioned above, into
a heparinized falcon vial (Corline Systems AB) and used
immediately.
2.4. The QCM technique and measurements
In brief, the QCM-D technique (Rodahl et al., 1995;
Hook et al., 1998; Voinova et al., 1999; Marx, 2003) is
based on a quartz crystal sandwiched between two gold elec-
trodes. When voltage is placed over the crystal, a lateral
oscillation starts with a fundamental frequency of 5 MHz.
The QCM-D also registers the frequencies at the third, fth
and seventh overtone (15, 25 and 35 MHz, respectively),
where the third overtone often was the most stable. When
molecules are adsorbed, the frequency (f) drops in a linear
proportion to the adsorbed mass (normally in the ng/cm
2
range) according to the Saurbrey equation (Saurbrey, 1959).
By switching the voltage on and off, a passive registra-
tion of the viscoelastic damping (D) can be performed. All
QCM-D measurements were obtained during no-ow condi-
tions.
2.4.1. QCM-D measurements of contact activated
coagulation
Citrated plasma (100 l) was added to a modied QCM
sensor surface placed in an open measurement cell (model
QWiC 301, Q-Sense, Gothenburg, Sweden) and incubated
for 5 min in order to achieve a stable baseline. A solution of
plasma (90 l) and CaCl
2
(10 l, 0.25 M) was mixed (30 s)
and added to the stabilized surface. The propagation of the
coagulation is observed as an initial lag time followed by a
frequency shift, which describes the kinetics of the coagu-
lation. After the coagulation is completed, the nal value of
the f and D can be analyzed to attain a description of the den-
sity of the formed clot. Time of onset, slope of the curve and
total coagulation time formed the basis of this analysis (see
Fig. 1).
82 M. Andersson et al. / Biosensors and Bioelectronics 21 (2005) 7986
Fig. 1. Graphs of representative coagulation experiments performed on test
surfaces. As indicated there are three parameters: (A) Onset time, measured
as time from addition of Ca
2+
to apparent frequency decrease. (B) Fibrin
deposition rate (slope of the curve at inection). (C) Total frequency shift at
plateau. Polymers used in this experiment are described in Table 2. Heparin
surfaces (Hep) and titanium surfaces (Ti) were also used. It was noted that
Hep surfaces measured no QCM-Dregistration of brin deposition or visual
coagulation. Each type of surface was tested at least three times without any
major differences.
2.4.2. QCM-D measurement of surface deposited C3
fragments
Serum diluted (1:5) in veronal buffered saline (VBS)
(0.15 M NaCl, 1.8 mM Na-5,5-diethylbarbiturate, 3.1 mM
5,5-diethylbarbituric acid, 0.5 mM NaN
3,
0.5 mM MgCl
2
with 0.15 mM CaCl
2
pH 7.4) was added to the modied
sensor surfaces which was placed in a temperature con-
trolled measurement chamber (22.0 0.2

C) (model QAFC
301, Q-Sense, G oteborg, Sweden). The serum was incu-
bated for 30 min. After rinsing with VBS (5 min), rabbit anti-
human C3c (DakoCytomation A/S, Glostrup, Denmark) was
added (1:50 in VBS, 20 min), followed by another VBS rinse
(5 min). The frequency shift produced by the added antibod-
ies was measure and used as an estimate of the degree of
complement activation (see inset Fig. 3). All QCM-D mea-
surements were performed in triplicates. In previous studies
(Sellborn et al., 2003) it has been shown that there are no
C3 fragments to be found on the titanium surfaces, although
the solution contains high levels of C3a (Hong et al., 1999a).
Hence, in this study, titaniumwas omitted fromC3 fragments
measurements. However, an upcoming study focusing on this
subject will be presented in the near future.
2.5. The slide chamber model and its measurements
The slide chamber model is a well-documented method
(Hong et al., 1999b) for studying mainly soluble end prod-
ucts of the complement system and the surface induced co-
agulation system. The model consists of two wells of PMMA
rings with a height of 5 mm and an inner diameter of 1.9 cm
xed to a PMMA microscope slide. Each well has a volume
of 1.65 ml. For this experiment, the wells were coated with
heparin and lled with 1.3 ml blood and 1 IU/ml soluble hep-
arin (Lvens Kemiske Fabrik, Ballerup, Denmark). Asecond
slide, the test surface, was placed on top. The whole construc-
tion was then sealed with two clips. O-rings were used be-
tween the wells and the test surface to prevent leaking. When
sealed, the construction was placed on a horizontally rotat-
ing disc in a water bath (37

C, 30 min). After incubation, the

chambers were opened and the blood was collected into two
heparinized vials containing EDTA (65 l, 0.2 M, pH 7.4). A
blood sample was taken prior to incubation and directly put
into two heparinized vials and used as control. The remaining
platelet count was measured in a cell counter (Coulter A
c
T
diff analyzer, Coulter Corporation, Miami, FL, USA). The
blood was then centrifuged (Minifuge T, 4000 rpm, 3450 g,
5

C, 25 min) and the cells discarded. The remaining plasma

was further analysed for C3a, TCC, TAT and TSP. These fac-
tors were measured with EIA standardized tests. The EIA
microtiter plates were analysed in a Multiskan MCC/340 mi-
crotiter reader at 492 nm(Labsystems, Vantaa, Finland). Two
slides for each surface modication were analysed, resulting
in four measurements per surface type. Heparin coated slides
were used as negative control.
2.5.1. Soluble C3a
Plasma diluted 1:100 in working buffer, PBS contain-
ing 1% (w/v) bovine serum albumin and 0.1% (v/v) Tween
20, was incubated in wells coated with mAb 4SD17.3
which served as capture antibody. As previously described
C3a was detected using biotinylated anti-C3a antibod-
ies followed by HRP-conjugated streptavidin (Nilsson et
al., 1988). Zymosan-activated serum (shown to contain
15,000 ng C3a/ml when compared to a calibrated solution
of puried C3a) served as standard. The values are given in
ng/ml.
2.5.2. Soluble TCC (sC5b-9)
The complement activation product sC5b-9 was measured
using a modication of the previously described method by
Mollnes et al. (1985). Plasma samples diluted 1:5 in work-
ing buffer were added to wells coated with anti-neoC9 mAb
McaE11, a kind gift from Prof. Tom-Eirik Mollnes, Uni-
versity of Troms, Norway. Polyclonal anti-C5 antibodies
(Dako, Denmark) diluted 1:500 in working buffer were used
for detection. Zymosan-activated serum, dened containing
40,000 arbitrary units per ml (AU/ml) served as standard.
2.5.3. Soluble thrombinantithrombin complex
Thrombinantithrombin complexes (TAT) were measured
using antibodies fromEnzyme Research Laboratories (South
Bend, IN, USA). TAT was captured in wells coated with anti-
human thrombin diluted 1:200. HRP-coupled anti-human an-
tithrombin antibody diluted 1:200 was used for detection. As
a standard puried thrombin mixed with excess of antithrom-
bin in the presence of heparin was used. The highest point in
the standard curve was 200 ng/ml TAT.
M. Andersson et al. / Biosensors and Bioelectronics 21 (2005) 7986 83
2.5.4. Thrombospondin (TSP)
Thrombospondin was measured using monoclonal an-
tibodies directed against TSP-1 (Immunotech S.A., Mar-
seille, France). As previously described by Bergseth et al.
(2000) clone P12 was used as capturing antibody and bi-
otinylated clone P10 as detecting antibody. Streptavidin
horseradish peroxidase conjugate (Amersham Biosciences,
Buckinghamshire, UK) was used to achieve staining. Nor-
mal serum was used as standard.
2.6. Statistical evaluation
Students t-test two-tailedandunequal variance was usedto
compare the results from the different surface modications.
3. Results
3.1. Coagulation
A summary of representative experiments on test surfaces
are shown in Fig. 1. We noted that there was a large span
in the results obtained from the different surfaces. As ex-
pected, the titanium surface (positive control) displayed the
fastest time of onset (A) as well as a very rapid brin depo-
sition rate (B). On the other hand, the total frequency shift
after coagulation was comparatively low for Ti. The heparin
grafted surface we used as control is an anti-coagulation sur-
face and displayed no changes in A, B or C, as expected. The
polymer surfaces displayed differences in all three parame-
ters (A, B and C). For the polymers the A and C parameters
appeared to be related, but the B parameter seemed to be in-
dependent of A and C. Numerical data from Fig. 1 are also
presented in Table 2 together with the results obtained from
the slide chamber model. In the QCM-D experiments we ob-
served that the two controls Hep and Ti, behaved as expected
concerning time to onset. Furthermore, parameters A and B
discriminated fully between the different polymer surfaces,
since the standard deviations were comparatively low, which
indicates good reproducibility. However, the results from the
slide chamber model (right hand side of Table 2) differed
between the Hep and Ti for TAT and remaining platelets
count but not for TSP, where the results were unclear. The
slide chamber model data for the polymer surfaces were hard
to interpret due to low reproducibility.
Table 2
Summary of the coagulation cascade as measured with QCM-D (onset time and brin deposition rate), EIA (TAT and TSP) and cell-counting (platelets)
Surface Onset time (A) (min) Fibrin deposition rate (B) (Hz/min) TAT (ng/ml) TSP (ng/ml) Platelets (% of control values)
Untreated 9 0 n.d. 100 1.0
Heparin No coagulation No coagulation 24 4.3 39 70 93 1.9
Titanium 15 0.58 144 33 51000 9000 82 12 0.36 0.14
PET 19 3.5 107 8.6 2700 3000 59 43 86 17
PE 19 1.7 43 2.7 8800 9300 80 22 58 18
PMMA 24 2.8 25 9.2 17 4.6 37 6.4 93 3.6
PDMS 20 8.8 38 20 34 5.9 40 4.3 88 2.9
Mean values and standard deviation (S.D.) are shown. The untreated samples had not been in contact with any surface.
Fig. 2. Representative f/D plots of the current surfaces. The frequency shift
(f) is given on the Y-axis, registration starts for all surfaces when Ca
2+
is
added (f =0). The corresponding dissipation values are given on the X-axis.
The experimental time span is 2 h. We interpret the result as the PET surface
inducing the clot with the highest density and PMMAwith the lowest density.
All plots appear linear except the PET plot, which is slightly curved.
As mentioned in the aim, we also set out to study the D
parameter in relation to clot formation. An established way
of studying the viscoelastic effect is to plot the frequency
shifts from coagulation kinetic experiments against D values
from the same experiments in a diagram. Hence, a relatively
low value of dissipation means higher rigidity. The f/D ratio
can be used as an estimation of the clot density. A high f
combined with a low D value would indicate a high density
clot. Graphs of f/D plots from the current surfaces are shown
in Fig. 2. As expected, the Hep plot is very short and has
no dened curvature. The Ti plot appears to illustrate the
formation of a low density clot. However, we believe that
this is a result of the clots low adsorption properties to the
Ti surface, i.e. the clot is not rmly attached to the surface
and slips over the surface leading to an underestimation of
mass. This may also explain the relatively low frequency
shift (C parameter), i.e. an underestimation of mass of Ti,
as seen in Fig. 1. The clots were always rmly attached
to the polymer surfaces. PDMS and PMMA displayed
a true low density clot while the PET surface showed a
high density clot. However, the clot formed on the PET
surface seemed to change during time from higher to lower
density.
84 M. Andersson et al. / Biosensors and Bioelectronics 21 (2005) 7986
Fig. 3. Results from complement activation on various test surfaces. Empty bars are TCC (AU/ml), grey bars represent amount C3a (ng/ml) and black bars
represent amount adsorbed anti-C3c (ng/cm
2
). Note that C3c measurements were not done on titanium. Inset: a graph of a representative C3c measurement
(PET surface). Human serum is added after a stable baseline is achieved. After incubation, the serum is rinsed off with VBS buffer and anti C3c is added. The
amount of adsorbed antibodies (ng/cm
2
) is calculated from the frequency shift (f) and taken as an arbitrary measure of surface bound complement fragments.
3.2. Complement activation
The principle of determination of complement activa-
tion with QCM-D is illustrated in the inset in Fig. 3. When
serum is added to a test surface, a deposited protein layer
will be formed that partly consists of complement factors.
After rinsing in buffer, the surface is incubated with anti-
complement factor 3 (C3c). The amount of deposited anti-
C3c antibodies is a good arbitrary measure of deposited
C3.
Adsorption of anti-C3c after serum incubation is shown
in Fig. 3 and is compared with results from the uid phase
reactants, C3a and TCC. All surfaces except Hep showed sig-
nicantly higher activation than the untreated control for both
C3a and TCC. Titanium, PE, PDMS and PMMAshowed sig-
nicantly higher activation than PET regarding TCC results.
Furthermore, PE showed signicantly higher activation than
PMMA (P<0.05, Students t-test, two tailed distribution,
equal variance). The QCM-D measurement of C3 fragment
is graded as follows: PETPEPDMSPMMAHep.
PE and PDMS showed signicantly higher amounts of C3
fragments than Hep (P<0.05, Students t-test, two tailed
distribution, equal variance). Note that sera incubated on
the PET coated surface resulted in the highest complement
activation amongst the polymers, while the EIA measure-
ments ranked PET as the least complement activating
surface amongst the polymeric test surfaces. Except this,
the internal correlations between the different surface
modications are good. Desorption was noted on the Hep
surface. Probably, the VBS buffer (containing anti-C3c)
washed off the serum proteins from the surface. The results
should be interpreted as a low degree of complement
activation.
4. Discussion
The results from the present study clearly indicate that the
QCM-D successfully can be used for detailed investigation
of both surface induce plasma coagulation and complement
activation. The methodology was found to be easy to use and
the reproducibility was high.
One of our most important nding was that surface asso-
ciated brin formation could be described with at least four
parameters using the QCM-D method; (1) time to onset of
clot formation, (2) rate of clot assembly, (3) total frequency
shift at stable plateau and (4) clot density. Few other meth-
ods, if any, can describe the surface induced clot formation
with so many parameters. The reason probably lies in the
fact that the clot analysis takes place directly on the modied
sensor surface. In contrast to most other traditional methods,
measurements are made separately from the surface. It has
previously been reported that time of coagulation onset cor-
relates with the amount of brin deposition (Sanchez et al.,
2002). Our ndings support this correlation, even though Ti,
with the shortest onset time, did not seem to have a partic-
ularly high brin deposition. The theory was also supported
by the results presented in Fig. 1. A new parameter was stud-
ied as well; the brin clot density, assessed in Fig. 2, where
the frequency shift was plotted against the dissipation. If this
has any implications for the integration of a biomaterial (e.g.
porous implants) or for wound healing cannot yet be estab-
lished. However, an intriguing nding was presented in an
article (Jorneskog et al., 1996) reporting that clots from pa-
tients with insulin dependent diabetes mellitus showed to
have a less porous brin network, i.e. a denser clot. There
have also been reports supporting that subjects suffering from
different variations of dysbrinogenemia have a denser b-
M. Andersson et al. / Biosensors and Bioelectronics 21 (2005) 7986 85
rin clot formation compared to healthy subjects. They also
showed decreased endothelial cell ingrowth. The decrease of
cell ingrowth was not related to RGD epitopes (Collen et al.,
2001). Both groups of patients have impaired wound healing,
although of different origin.
Considering the complement activation system, there was
a good correlation between QCM-D and C3a and TCC deter-
minations. This was surprising, since complement activation
was determined in serum with the QCM-D method, whereas
C3a and TCC were determined in whole blood in the slide
chamber. An inconsistent observation, however, was the dis-
crepancy in the PET surface measurements. The EIAmethod
ranked PET as having the lowest complement inducing prop-
erties of the polymers, while QCM-Dranked PETas the high-
est complement inducing surface. Per denition, the molar
amount of C3 fragments can never be more than that of C3a.
One explanation of the inconsistency could be that both C3a
and TCC adsorbs to the protein/platelet fouled PET surface.
An alternative explanation could be that native C3 associates
to the PET surface without being cleaved, hence, there would
be low amount of C3a, but high response in C3 as measured
with the QCM-D.
A general observation when comparing the slide chamber
model and the QCM-D was that the correlation was high in
the complement experiments but low in the coagulation ex-
periments. The lowcorrelation in blood coagulation is proba-
bly due to the biological complexity of the blood coagulation
system, involving both soluble proteins and cells. There is
also another important factor that could offer an explanation;
QCM-D measures the phase transition when soluble brino-
gen forms a solid brin clot, which is completely different
from measuring a single type of protein in solution. The dif-
ferences in brin clot formation rate, time of onset and the ar-
chitecture of the formed clot did not seem to relate to platelet
loss, neither did TSP or TAT.
The activation of the complement system however, in-
cludes a central activation mechanism, the cleavage of C3.
The appearance of factors like C3a, C3b, C3c, C5a and TCC
will quantitatively depend on the central activation mecha-
nism. In general, good correlation was therefore expected in
the complement activation experiments.
5. Conclusion of major ndings, limitations, future
studies
It may be concluded that the QCM-D methodology pro-
vides a simple and trustworthy way to study parameters at
the interface between blood components and solid surfaces.
It also broadens the possibilities in the biocompatibility re-
search area by introducing a direct way of measuring the clot
density of surface induced coagulation. It is generally as-
sumedthat invitrostudies of newmaterials have lowprognos-
tic values regarding in vivo trials. However, we hope that fur-
ther studies using the QCM-D methodology will shed more
light on the bio-interface research eld.
Future studies may include a more detailed investigation
of the structure of the different brin clots, perhaps by using
confocal microscopy and/or AFM.
Acknowledgements
We wish to thank Entic medical systems AB, G oteborg,
Sweden, for nancial support.
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