Journal of Colloid and Interface Science 287 (2005) 35–42

www.elsevier.com/locate/jcis

Enzyme immobilization on poly(ethylene-co-acrylic acid)

films studied by quartz crystal microbalance
with dissipation monitoring
Xiaodi Su a,∗ , Yun Zong a , Ralf Richter b , Wolfgang Knoll a,c
a Institute of Materials Research and Engineering, 3 Research Link, 117602 Singapore
b Q-Sense AB, Redegatan 13, 42677 Västra Frölunda, Sweden
c Max-Planck-Institut für Polymerforschung, Ackermannweg 10, 55128 Mainz, Germany

Received 3 December 2004; accepted 24 January 2005

Available online 10 March 2005

Abstract
In this study, we use the quartz crystal microbalance with dissipation monitoring (QCM-D) to study the immobilization of the enzyme
horseradish peroxidase (HRP) on poly(ethylene-co-acrylic acid) (PEAA) films. The surface polarity of spin-coated PEAA films was varied
by heat treatments in air or in a 30% NaOH aqueous solution leading to COOH-depleted or COOH-enriched surfaces, respectively. Two
reaction schemes, direct adsorption and amine coupling, were employed for HRP immobilization on the two surfaces. The shifts in frequency
and dissipation,
f and
D, measured by QCM-D and the ratio
D/
f were used to evaluate the binding amount and the conformation
of the adsorbed enzyme. It is found that HRP immobilized via covalent linkages forms rigid and little dissipative films. In contrast, directly
adsorbed HRP films exhibit a highly dissipative structure. HRP-catalyzed oxidation of the 4-chloro-1-naphthol in the presence of H2 O2 was
used to characterize the catalytic activity of the HRP films. The results show that the enzymatic activity of the covalently immobilized HRP
tends to be higher.
 2005 Elsevier Inc. All rights reserved.

Keywords: QCM-D; Polyethylene-co-acrylic acid; HRP

1. Introduction
Quartz crystal microbalance (QCM) is a well-established
noninvasive technique suitable for the investigation of ad-
sorption/desorption processes on solid surfaces [1]. The
merits of the QCM technique are the simplicity and sensitiv-
ity, by which a wide range of interfacial adsorption reactions
can be monitored, on a variety of supports, in real time. For
rigid, ultrathin, and evenly distributed adsorbed layers, the
Sauerbrey equation [2] describes successfully the propor-
tional relationship between the adsorbed mass (
m) and the
shift of the QCM crystals’ resonance frequency (
f ). On the
other hand, if the adsorbed material exhibits a viscoelastic
* Corresponding author. Fax: +65-68720785.
E-mail address: xd-su@imre.a-star.edu.sg (X. Su).
0021-9797/$ – see front matter  2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.jcis.2005.01.089
behavior, such as for layers of proteins [3] or DNA [4], sub-
stantial deviations from the Sauerbrey equation can occur,
depending on how the oscillatory motion of the crystal prop-
agates into and through the adsorbed viscoelastic films [5].
The damping behavior of the QCM crystal is related to the
conformational or structural properties of the viscous lay-
ers.
One way to study the damping effect on a quartz sensor
is to measure the dissipation D (inversely proportional to
the Q factor) of a nondriven (freely oscillating) crystal [6].
The so-called QCM-D is such an instrument that measures
changes in frequency (
f ) and dissipation (
D) simulta-
neously. This is achieved by periodically switching off the
driving power and by recording the decay of the damped
oscillation. The time constant of the decay is inversely pro-
portional to D and the period of the decaying signal gives
36 X. Su et al. / Journal of Colloid and Interface Science 287 (2005) 35–42
the frequency f . The QCM-D technique has been success-
fully applied for studies of protein adsorption [7–10], protein
cross-linking [11,12], DNA hybridization [4,13], and many
other interfacial processes [14–16] on a variety of surfaces
(e.g., metals, SiO2 , polymers, and lipid membranes). The
combined
f and
D data analysis provides information
about the effective thickness, conformational changes, vis-
coelastic properties, and the hydration state of thin films of
macromolecules [1,3].
Enzyme immobilization on polymeric materials has been
utilized in many fields, including bioseparation, biosens-
ing, and biocatalysis. The immobilization strategies include
physical adsorption, entrapment, and covalent binding. The
stability and activity of the immobilized enzyme are deter-
mined by the changes of chemical nature/conformation of
the immobilized enzyme that are influenced by the reac-
tion schemes involved and the microenvironment provided
by the supporting material [17]. A detailed understanding of
the mechanisms underlying enzyme immobilization and ad-
sorption is a challenging scientific issue, having important
implications in designing suitable immobilization schemes
for a strong enzyme attachment with well-retained enzy-
matic activity.
In order to achieve covalent enzyme immobilization on
polymer substrates, efforts have been made to introduce
a variety of functional groups into the polymer materials.
Grafting acrylic acid monomers on polymer substrates, for
example, is an effective method [18–20], by which enzymes
can be immobilized via amide bond formation (support–
co-NH–enzyme). Recently, the use of polymers containing
acrylic acid residues as substrates for covalent tethering of
biomolecules has also been reported [21–23]. Poly(ethylene-
co-acrylic acid) (PEAA), for example, is a polymer with
a nonpolar polymer scaffold containing a small amount of
a polar groups. In previous studies, bulk PEAA films [22]
and thin PEAA coatings [23] were used as solid supports
for covalent tethering of alkylamino-modified DNA for hy-
bridization analysis.
In this study we use the QCM-D technique to investigate
the immobilization of the enzyme horseradish peroxidase
(HRP) on PEAA surfaces. PEAA thin films spin-coated
on quartz disks were heated in air or in aqueous sodium
hydroxide in order to modulate the surface’s polarity and
hydrophobicity [24]. HRP was immobilized either by non-
specific adsorption to the PEAA surfaces or through amine
coupling, i.e., via EDC/NHS activation of the surface COOH
groups. The QCM-D responses, i.e.,
f and
D, were
employed together with the
D/
f ratio to evaluate the
amount of bound protein and the conformation of the ad-
sorbed enzyme. Theoretical modeling of the data was carried
out using a Voigt-based representation to deduce the effec-
tive thickness, viscosity, and elasticity of the enzyme films.
Using the combined response of frequency and dissipation,
we characterize here the formation of a precipitate upon
catalytic oxidation of 4-chloro-1-naphthol (4-CN) by immo-
bilized HRP.
2. Experimental
2.1. Materials
Poly(ethylene-co-acrylic acid) (20% w/w acrylic acid)
was purchased from Aldrich (St. Louis, MO). Peroxidase
type (EC 1.11.1.7) from horseradish (HRP, 10,000 units/mg
solid), N -ethyl-N  -(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC), and N -hydroxysuccinimide (NHS)
were purchased from Sigma (St. Louis, MO). PBS buffer
(pH 7.2) was used for HRP immobilization. Hydrogen per-
oxide, 4-chloro-1-naphthol (10 mM solution in diethylene
glycol), and 20 mM Tris buffer (pH 7.5) were from a HRP
Conjugate Substrate Kit (Bio-Rad Laboratories, CA). The
substrate solution was 2 mM 4-CN diluted using the Tris
buffer with the presence or absence of H2 O2 .
2.2. QCM-D measurement
The QCM-D (Q-Sense, Gothenburg, Sweden) technique,
described in detail elsewhere [25], is an extension of the tra-
ditional QCM technique in the time domain. It measures

f and
D by periodically switching off the driving cir-
cuit and recording the decay of the damped oscillation, and
5 MHz, AT-cut crystals with gold electrodes were used as
reaction carriers. These crystals provide a mass sensitivity
of 17.7 ng cm−2 Hz−1 . During the measurement, the crys-
tal was mounted in a thermostated liquid chamber, designed
to provide a rapid, nonperturbing exchange of the liquid
in contact with one side of the sensor. This system allows
for the measurement of up to four harmonics. In this study,
the frequency and dissipation responses were recorded at
around 15, 25, and 35 MHz, corresponding to the overtones,
n = 3, 5, and 7, respectively. For clarity, only the normalized
frequency shifts,
fnormalized =
fn /n, and the dissipation
shifts,
D, for the third overtone are presented. Data at
other overtones are included for the modeling of the effective
thickness and viscosity using the Voigt-based representation
[1,4].
2.3. Preparation of PEAA coatings on sensor disks
PEAA thin films were spin-coated on one side of the sen-
sor disks from a 5 mg/mL THF solution at 2500 rpm. The
resulting disks were heated at 80 ◦ C in either a 30% NaOH
aqueous solution for 30 min or in air for 1 h to achieve PEAA
surfaces enriched or depleted in COOH, respectively. The
surface COOH concentrations were determined by using the
toluidine blue O (TBO) method [19,20]. In brief, the COOH
concentration is determined by the amount of cationic dye
adsorbed on the polymer surface through complexation with
the COO groups at pH 10.
2.4. HRP immobilization
Two reaction schemes were involved for HRP immobi-
lization. In one scheme, the PEAA-coated sensor crystal
 X. Su et al. / Journal of Colloid and Interface Science 287 (2005) 35–42
surface was first treated with a 0.4 M EDC and 0.1 M NHS
aqueous solution (1:1 mixture) for 7 min. After this treat-
ment, the surface was equilibrated with PBS buffer and the
HRP solution (0.5 mg/mL in PBS) was then applied. After
binding equilibrium was reached, the cell was rinsed with
PBS buffer to remove loosely attached enzyme. In the other
scheme, the HRP solution was directly applied on the PEAA
surface calibrated in PBS buffer, without EDC/NHS activa-
tion.
2.5. Catalytic activity of the bound HRP
HRP-catalyzed oxidation of 4-CN in the presence of
H2 O2 was used to study the catalytic properties of the immo-
bilized HRP. Surfaces with immobilized HRP were exposed
to a 4-CN substrate solution in the absence of H2 O2 to cal-
ibrate the baseline. After equilibrium, the same substrate
solution containing 7.5 × 10−3 mM of H2 O2 was injected
to react. The precipitation of oxidized 4-CN on the sensor
surfaces was followed by QCM-D.
3. Results
3.1. Surface preparation and HRP immobilization
PEAA is an organic polymer with a nonpolar scaffold
containing a small amount of polar carboxyl groups. Heat-
ing the polymer in air or in aqueous sodium hydroxide
will change the surface polarity due to the diffusion of
the functionality between the surface and the bulk of the
polymer [24]. Fig. 1 depicts the process of the change in sur-
face composition of spin-coated PEAA films on the sensor
crystal. Upon heating in air, the polymer surface becomes
depleted in COOH groups, while heating in 30% NaOH
leads to a COOH-enriched surface. Using the TBO dying
method, we confirmed that the surface COOH density for the
COOH enriched surfaces is 2–3 times of that of the COOH-
depleted surfaces. The corresponding polarity indicated by
the advancing contact angles is 92.0 and 68.5◦ , respectively.
EDC/NHS-based amine coupling is a frequently used
method for the covalent tethering of proteins onto COOH-
containing substrates. EDC/NHS activates the COOH groups
to form N -hydroxysucciniimide esters. The latter promote
the formation of amide bonds between the COOH groups on
Fig. 1. Schematic illustration of the diffusion of carboxylic acid groups between the surface and the bulk of the PEAA films spin-coated on the sensor crystals
upon heating treatments.
37
the substrate and the primary amine of the protein to be im-
mobilized [26]. The first part of Fig. 2 shows the QCM-D
response for the EDC/NHS activation of a COOH-depleted
surface. The application of EDC/NHS (2 min) after cali-
bration of the initial baseline in PBS results in significant
shifts in frequency and dissipation, which are almost entirely
reversed after replacing the EDC/NHS solution with PBS
buffer. This response likely reflects the difference in viscos-
ity and density between PBS buffer and EDC/NHS aqueous
solution and is thus not surface related. The changes in the
adsorbed mass associated to the formation of N -hydroxy-
succiniimide ester are expected to be small [27], which
is indeed reflected by the minor net changes in f and D
measured after rinsing in buffer solution. The successive ap-
plication of HRP on this activated surface (Fig. 2, 13 min)
resulted in a steady frequency decrease accompanied with
a dissipation increase over time, which reflects the adsorp-
tion of the enzyme at the sensor surface.
f and
D at
saturation were −22 Hz and 0.8 × 10−6 , respectively. The
resulting ratio |
D/
f | (the induced energy loss per cou-
pled unit mass) is 0.04 × 10−6 Hz−1 .
The same immobilization reaction was repeated on the
COOH-enriched surface. The EDC/NHS activation results in
only minor net changes in f and D (data not shown) as in the
COOH-depleted case. Successive exposure to HRP (Fig. 3)
results in
f = −17 Hz and
D = 0.7 × 10−6 that leads
to |
D/
f | = 0.04 × 10−6 Hz−1 . These values are similar
to those obtained on the COOH-depleted surface, indicating
that the amount of HRP binding and the protein conforma-
tion on both types of PEAA surfaces are similar.
In another reaction arrangement, HRP was directly ap-
plied onto PEAA surfaces that were not activated by EDC/
NHS. Figs. 4 and 5 show the QCM-D responses for the
COOH-depleted and COOH-enriched surfaces, respectively.
We note that both the frequency shifts (−36 Hz versus
−25 Hz) and the |
D/
f | values (0.16 × 10−6 Hz−1 ver-
sus 0.08 × 10−6 Hz−1 ) at saturation differ for these two
surfaces. This suggests a profound influence of the COOH
density on the protein adsorption, in contrast to our observa-
tions on the EDC/NHS-activated surfaces.
If comparing between immobilization with and without
EDC/NHS activation, adsorption proceeds generally slower
on nonactivated surfaces, but leads to higher frequency shifts
and higher dissipation changes. The |
D/
f | values are
always higher for the physically adsorbed HRP on each in-
dividual surface. Table 1 summarizes the QCM-D signals
38 X. Su et al. / Journal of Colloid and Interface Science 287 (2005) 35–42

Fig. 2. HRP immobilization on COOH-depleted surface through EDC/NHS activation.

Fig. 3. HRP immobilization on EDC/NHS-activated COOH-enriched surface (EDC/NHS activation is not shown).

Fig. 4. HRP adsorption on COOH-depleted surface through direct adsorption.

(
f,
D, and |
D/
f |) at saturation for HRP bound to thickness and viscosity of the deposited HRP films through
the two surfaces using the two reaction arrangements. fitting using a Voigt-based model [4]. The “effective” values
In addition to the qualitative observations of the HRP describe the entire film composed of water and HRP. The fit-
immobilization behavior, we have quantified the effective ted parameters are based on a HRP density of 1.15 g/cm3 (an
 X. Su et al. / Journal of Colloid and Interface Science 287 (2005) 35–42
Fig. 5. HRP adsorption on COOH-enriched surface through direct adsorption.
Table 1
Summary of the QCM-D data (
f and
D at saturation) for HRP immobilization on COOH-enriched and COOH-depleted PEAA surfaces under different
reaction schemes
COOH-enriched surface
+ EDC/NHS

f (Hz) 17

D (×10−6 ) 0.7

D/
f (×10−6 ) Hz−1 0.04
Thickness (nm) 3.7
Mass (ng/cm2 ) 426
Viscosity (×10−3 N s/m2 ) 6 3.6
average of 1.0 g/cm3 for water and 1.3 g/cm3 for the pro-
tein) [7–9].1 The fitted results (Table 1) show that the HRP
films, formed on the EDC/NHS-activated surfaces, have a
higher viscosity. The resulting effective thicknesses, d ≈
4.1 nm (COOH-depleted surface) and d ≈ 3.7 nm (COOH-
enriched surface), are similar to a previously reported HRP
thickness of 3.6 ± 1.9 nm (tapping mode AFM data), where
the HRP molecules were trapped on a dithiobis-N -suc-
cinimidyl propionate-treated gold surface using a similar
covalent attachment chemistry [28].
3.2. The catalytic activity of immobilized HRP
HRP is known to catalyze the oxidation of 4-CN in the
presence of H2 O2 leading to the precipitation of the oxide
in diethylene glycol containing solution. In this study we
use this precipitation reaction to evaluate the catalytic ac-
tivity of surface-bound HRP by exposing the surface to a
mixture of 4-CN and H2 O2 . As an example, Fig. 6 shows
the corresponding QCM-D measurement, where the HRP
1 The effect of the choice of the HRP density on the fitted viscosity and
the mass is negligible. The fitted thickness varies reversely proportional
with changes in the input density [3]. The error is, however, expected to
be smaller than ±15% as the physically reasonable range for the density
(1.0 to 1.3 g/cm3 ) is narrow.
39
COOH-depleted surface
No EDC/NHS + EDC/NHS No EDC/NHS
25 22 36
2.1 0.8 5.7
0.08 0.04 0.16
5.1 4.1 6.2
587 472 713
6 2.9
immobilization is achieved using direct adsorption on a
COOH-depleted surface. When the QCM-D sensor carrying
the HRP layer was exposed to the HRP substrate solu-
tion (4-CN in Tris/diethylene glycol buffer in the absence
of H2 O2 ), the change in viscosity of the liquid medium
resulted in a drop in f and an increase in D (data not
shown). Subsequent coincubation of 4-CN and H2 O2 in the
same buffer led to substantial changes in f and D (Fig. 6),
which are indicative of the formation and deposition of oxi-
dized 4-CN. At saturation,
f = 264 Hz, and |
D/
f | =
0.067 × 10−6 Hz−1 are reached. The initial rate of the sub-
strate deposition is 2.7 Hz/s.
Qualitatively similar responses are obtained for all condi-
tions of HRP immobilization, confirming that the deposited
proteins are enzymatically active. For covalently bound HRP
on EDC/NHS-activated, COOH-enriched PEAA, the final
values of
f = 342 Hz and
D/
f = 0.063 × 10−6 Hz−1
are obtained. The initial rate of the substrate deposition
is 4.0 Hz/s, being higher than that with the directly ad-
sorbed HRP. The comparison of the initial rates of pre-
cipitate deposition [29] reveals that the covalently bound
HRP has a stronger activity. In addition, it is notable that
the |
D/
f | values are similar for all employed HRP-im-
mobilization protocols. This indicates that the precipitate
layers exhibited identical viscoelastic properties. The differ-
40 X. Su et al. / Journal of Colloid and Interface Science 287 (2005) 35–42
Fig. 6. Enzyme-catalyzed oxidation of 4-CN in the presence of H2 O2 . The enzyme-immobilized COOH-depleted surface (via direct adsorption) is exposed to
the HRP substrate solution (4-CN in Tris buffer with a small amount of diethylene glycol) to create a response baseline. Once this response is stabilized, the
substrate solution is replaced by the same solution containing H2 O2 .
ences in
f observed for the precipitation reaction represent
thus true differences in the amount of adsorbed precipitate.
4. Discussion
We have investigated the binding behavior of HRP on
PEAA surfaces via different immobilization strategies. As
evidenced by Table 1 and Figs. 2–5, the HRP adsorption
on the PEAA surfaces using different immobilization strate-
gies exhibits remarkable differences in the binding kinetics
and saturation behavior (
f ,
D, and |
D/
f | ratio).
These differences are believed to be related to the amounts
of bound protein, the binding mechanisms, and the structure
of the formed protein films and will be discussed below.
According to the general understanding of the behavior
of a macromolecular film, e.g., adsorbed proteins, as sensed
by QCM-D, water is known to be trapped and to be sensed
as additional mass [7–10]. The amount of trapped water can
vary significantly depending on the structure of the formed
protein film [3], and an increased water content is usually
correlated with increased flexibility, i.e., a larger |
D/
f |
ratio [3,30]. Based on this understanding, we interpret our
observation of the smaller |
D/
f | values for the HRP pro-
teins adsorbed to EDC/NHS-activated surfaces as a strong
indication that the adsorbed films are more rigid (contain-
ing less water) than directly adsorbed films. In agreement,
the adsorption on EDC/NHS-activated surfaces results in
protein films of higher viscosity and lower thickness (Ta-
ble 1). The COOH density did not significantly influence the
film structure (
D/
f ) and the adsorbed amount (
f ) of
these surfaces. Considering the size of the HRP molecule
(3.5 × 6.0 × 7.5 nm) [31], it appears that the number of sites
that is available on surfaces with the lower COOH density
is sufficient for tight attachment of a complete monolayer
of HRP. As such, additional binding sites on the COOH-
enriched surfaces have no further effect on the binding
behavior.
Compared to the immobilization on EDC/NHS-activated
surfaces, direct adsorption is observed to proceed more
slowly. It appears that in order for physisorption to take
place, an activation barrier must be overcome. Once ad-
sorbed, however, the enzyme is immobilized irreversibly. In
addition, directly adsorbed proteins exhibited an adsorption
behavior with rather high dissipation, indicating the forma-
tion of a water-rich and soft film structure. The fitting results
(Table 1) reveal that these HRP films are thicker and exhibit
lower viscosity than the HRP films adsorbed on EDC/NHS-
activated surfaces. Unlike the case of EDC/NHS-activated
surfaces, the surface polarity significantly influences the vis-
coelastic state (|
D/
f |) and the total adsorbed mass.
It is known that a native protein is usually covered
with hydrophilic residues (e.g., carboxyl, amino, and thiol
groups). At a pH value near the pI value, the protein tends to
retain its native structure without exposing the hydrophobic
residues [32]. This is exactly the case for the HRP in PBS
buffer (pH 7.2) in this study (pI of the HRP is 7.2 [19]).
Thus the direct adsorption of HRP to the PEAA surfaces
(Figs. 4 and 5) is believed to be dominated by polar–polar
interactions (some electrostatic interactions may also be in-
volved as the surface COOH groups are partially ionized at
pH 7.2 [22]). We suggest that the increased polarity of the
COOH-enriched surface leads to more extensive polar–polar
interactions between the protein and the surface, thus result-
ing in a more efficient HRP adhesion on the surface, being
detected as a layer with a smaller flexibility (|
D/
f | =
0.08 × 10−6 Hz−1 ) if compared to the COOH-depleted case
(|
D/
f | = 0.16 × 10−6 Hz−1 ).
From Figs. 2–5, the adsorption-induced dissipation shifts
seem, in both reaction schemes, to follow the frequency
shifts (cf., mass uptake), but not exactly. As examples,
Figs. 7 and 8 show the plot of
D versus
f for the immo-
 X. Su et al. / Journal of Colloid and Interface Science 287 (2005) 35–42
Fig. 7.
f vs
D plot for the HRP EDC/NHS immobilization on COOH-
enriched surface (data from Fig. 3).
Fig. 8.
f vs
D plot for the HRP direct adsorption on COOH-depleted
surface (data from Fig. 4).
bilization of HRP on EDC/NHS-activated, COOH-enriched
surfaces (data from Fig. 3) and for the direct immobilization
on COOH-depleted surfaces (data from Fig. 4), respectively.
These
D–
f plots eliminate time as an explicit para-
meter and as concluded in previous studies, the absolute
slope and the changes in the slope in these plots provide
information about the kinetic regimes and conformational
changes [33]. In the present cases, this is manifested by
the fact that the direct adsorption of HRP displays a linear
regime in the
D–
f plot, whereas the EDC/NHS-coupled
HRP shows two clearly distinguishable linear regimes, with
the initial part having a smaller slope and the second one
being significantly larger. Our interpretation is that in the
direct adsorption reaction, the proteins experience no con-
formational change at different surface coverage; while in
the amine coupling reaction, the protein molecules initially
reaching the surface form a rigid, presumably denatured pro-
tein film, which is followed by binding of protein in a more
flexible and native-like conformation. It is speculated that
it is the slight change in the conformation that leaves the
redox center exposed and becomes more easily accessible.
As a result, more reactivity is detectable for the covalently
bound HRP.
Enzyme-catalyzed chromogenic precipitations have been
used for signal amplification in surface analytical device-
41
based bioassay (e.g., QCM immunoassay [34,35], QCM
enzymatic assay [29,36,37], and ellipsometry immunoas-
say [38]). In the QCM studies, the deposition of the precipi-
tation is measured using the frequency shifts only, neglecting
the possible effect of structure and solvent content of the
precipitate layer on the frequency response. Our measure-
ments with simultaneous acquisition of f and D strongly
indicate that provided identical reaction conditions are used,
the measured frequency shifts can indeed be used for a rel-
ative comparison of the enzymatic activity. However, our
results also demonstrate that the precipitating film is soft
(|
D/
f | ∼ 0.06 × 10−6 , similar to the HRP films) and
hence solvent rich. This implies that the frequency response
may be strongly influenced by the solvent contained in the
adsorbed film. Consequently, the nature of the solvent may
significantly affect the frequency response and measure-
ments performed with different solvents are thus not a priori
comparable. In this respect the supplementary information
provided by the dissipation helps in validating the compa-
rability of frequency shifts and investigating the structural
properties of the adsorbed film.
5. Conclusion
We have demonstrated the use of the QCM-D tech-
nique for a detailed enzyme immobilization study. By using
different reaction schemes, the enzyme was immobilized
on PEAA films with different mechanisms. The combined
acquisition of frequency changes and dissipation changes
allowed for the detection of differences in the binding be-
havior, in the conformation, and in the activity of the bound
enzyme. This information is considered important in the de-
sign of suitable immobilization schemes that ensure a high
binding stability and maximized catalytic activity.
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