FACILE SURFACE MODIFICATION FOR COVALENT IMMOBILIZATION
OF ENZYMES ON DIFFERENT GEOMETRIES
Anna Cifuentes1, Laia Masramon1,3, Antoni Planas2 and Salvador Borrós1*
1
Grup d’Enginyeria de Materials (GEMAT), Institut Químic de Sarrià, Universitat Ramon LLull
Via Augusta 390 08017 Barcelona, [email protected]
2
Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon LLull
Via Augusta 390 08017 Barcelona
3
CETEMMSA Technological Centre, Jaume Balmes 37-39 08301 Mataró (Barcelona)
Abstract: Since the immobilization of enzymes on solid supports have been
presented as a challenging solution in many fields of biotechnology, we have
developed a novel method to covalently attach the enzyme 1,3-1,4-β-Glucanase
on polystyrene (PS) surfaces of flat and spherical shapes, which remains its
activity. The substrates were modified by Ar plasma-grafting with the monomer
pentafluorophenyl methactylate (PFM) and were analyzed by water contact
angle, Atomic Force Microscopy (AFM) and fluorescence microscopy. After
enzyme immobilization was assessed and monitored by the Quartz Crystal
Microbalance with Dissipation (QCM-D) technology, the activity of the anchored
enzyme was tested by using the dinitrosalicyclic acid (DNS) method. The
enzyme retains its activity after immobilization demonstrating the suitability of
the technique and providing a novel method of enzyme immobilization under
mild and controllable conditions.
Keywords:
Enzyme
Immobilization,
Plasma-Grafting,
Pentafluorophenyl Methacrylate, 1,3-1,4-β-Glucanase
1. Introduction
Immobilization of biological active species is
crucial for the fabrication of smart bioactive
surfaces.1-3 Up to now, a great variety of
methodologies have been described in order to
attach biomolecules on different substrates without
affecting its activity.1,2,4,5
For this purpose, plasma assisted treatment has
frequently been used to modify the surface nature
without affecting the bulk properties of the
material.5,6 Thus, it is possible to create materials
with a thin polymeric film on the surface able to
promote the anchoring of some kind of compounds.5
The monomer used in this work is the
pentafluorophenyl methacrylate (PFM), a reactive
acrylate-derived monomer that when is polymerized
or grafted on a surface offers a labile ester group
which is of a great interest owing its high reactivity
toward amino-terminated molecules and it has
QCM-D,
widely used for this purpose by our research
group.5,9-10
2. Experimental Section
Plasma
reactor.
The
plasma-grafting
modification was carried out with a home-­‐built
plasma reactor (Figure 1). Plasma treatment was
performed by using an excitation frequency of 13.56
MHz. Gases that were fed through the system pass
through a glass, CO2/acetone cooled trap for
collection of excess reactant before reaching the
pump (Edwards RV12 903). An analogical Pirani
type vacuum meter (MKS, USA) was connected
near the middle of the reactor chamber, to monitor
the reaction pressure. In a home built system, the
pulse generator controlled the pulsing of the radio
frequency signal, which was amplified by a 150 W
amplifier and passed via an analogue wattmeter and
a matching network to a 10 cm long coil located
around the exterior of the reactor. The typical base
pressure prior to all experiments was 2x10-2 mbar.
PFM monomer vapor was introduced at a constant
pressure of 1.5x10-1 mbar via a needle valve. The
reactor’s inner volume is approximately 3 L, while
the effective plasma volume is about 1.7 L.
Figure 1: Schematic diagram of the plasma reactor and its electrical
components
Plasma-grafting modification. Polystyrene beads
(Goodfellow; 900 µm diameter; PSb) were modified
with pentafluorophenyl methacrylate (Apollo
Scientific; PFM) by plasma grafting by using the
plasma reactor previously described. The substrates
were firstly activated by argon plasma, (5.0) which
was inserted to the reactor in a constant flow of
7.5x10-2 mbar. The continuous RF power setting was
15 W and was carried out during 15 min. After
surface activation, the plasma was turned off and the
Ar gas is closed. PFM monomer flask was then
opened until reaching the desired pressure during 15
min. This procedure was carried out three times
shaking the samples in between each treatment in
order to achieve a homogeneous modification along
the bead surface (PSb-PFM). When the grafting
procedure was finished, samples were removed from
the reaction chamber and stored until further use.
Modified surfaces characterization. The
modified surfaces were characterized by water
contact angle (DSA100, Krüss; WCA), Atomic
Force Microscopy (XE 100 Park System; AFM) and
fluorescence microscopy (AxioVs40, Zeiss Imaging
Solutions). In order to analyze the surface
modification by WCA and AFM, the plasma
grafting procedure was carried out on flat PS
substrates. The fluorescent assay was directly
performed to the modified PS beads by using
fluorescein-5-tiosemicarbazide (Fluka; FTSC) as a
fluorescent dye.
Enzyme synthesis. The enzyme used in this study
was the 1,3-1,4-β-glucanase. It was synthesized,
purified and quantified as previously described.7,8
Enzyme immobilization. The immobilization was
carried out in a 96 well plate that was previously
tested and did not give adsorption in the further
working λ. 20 units of PSb-PFM were incubated
with 10 nM 1,3-1,4-β-glucanase solution in 50 mM
phosphate buffer (Sigma-Adrich; PB) at pH 7. The
mixings were left in mild agitation during 1h at 4oC.
The total volume of the wells was removed and the
PSb with the immobilized enzyme (PSb-PFM+Gln)
were subsequently cleaned with PB and milli-Q
water. The same procedure was carried out with
original PSb without modification (PS+Gln)
QCM-D analysis. Quartz Crystal Microbalance
with Dissipation was used to characterize the
immobilization process of the enzyme to a PS sensor
modified with PFM at same conditions as the PSb.
The baseline was obtained with the PB 50 mM and a
30 µg/mL 1,3-1,4-β-glucanase solution was used to
study the immobilization. A final cleaning with 10
mM SDS solution and PB ensures a covalent
immobilization of the enzyme to the surface. The
viscoelastic properties of the resulting enzymatic
layer were studied in detail. The enzyme affinity
toward the PS sensor (PS+Gln) was checked as a
comparative control. The flow was fixed at 50
µL/min during the whole process.
Enzymatic activity. The enzymatic activity of the
immobilized enzyme (PSb-PFM+Gln and PS+Gln)
was tested using the DNS method. The samples and
controls were incubated with the enzyme substrate, a
solution of 1,3-1,4-β-glucane (Meganzyme) 4
mg/mL in PB 50 mM and CaCl2 0.1 mM, during 5
min at 55oC. A fraction of each of these mixtures
was added to the 3,5-dinitrosalicylic acid (DNS)
reagent solution. To obtain the color change required
to detect the different amounts of reducing sugar the
samples were boiled at 100°C for 10 min, cooled
down on ice and measured at 540 nm against a blank
with milli-Q instead of enzyme. The activity of the
free 1,3-1,4-β-glucanase (10 nM) synthesized was
quantified following the same protocol.
A
B
3. Results and Discussion
Plasma grafting modification. Plasma grafting
of PFM to PS substrates led to a considerable
decrease in contact angle of the original PS. The
later had a contact angle of 83o, which decreased to
52o after modification. This was an unexpected value
considering the hydrophobicity observed on the
PFM films polymerized by PECVD showed in other
work.5,9,10 These studies have proved that the
fluorinated ring from the PFM reminds exposed to
the surface, giving the hydrophobic behavior noticed
when it is polymerized in this conditions.
Nevertheless, when the PFM is grafted on the
polymeric surface by plasma grafting forms a
polymer brush layer, as can be interpreted from the
AFM images taken (Figure 2).
Figure 3: PSb functionalized with FTSC. (A) Original PSb incubated
with a FTSC solution, which shows no fluorescence. (B) Fluorescence
observed after the reaction of the FTSC with the PFM previously grafted
on the surface.
Enzyme immobilization. The immobilization of
the enzyme on the PSb-PFM was monitorized in
parallel with the QCM-D technology by using a PS
quartz crystal sensor (Figure 4).
Figure 4:QCM-D plot of the immobilization process and subsequent
cleaning. The baseline was obtained by the PB solution before the 1,31,4-β-Glucanase was introduced into the chamber to react with the
modified PS sensor. By cleaning with PB and SDS a covalent
immobilization was proved.
Figure 2: AFM images of the polymer brushes of PFM formed by
plasma grafting. (A) 2D image and (B) topographic profile
As a result of the formation of these polymeric
brushes, the coating does not cover the total surface
causing the decrease in hydophobicity observed.
Thus, the grafted surface may then provide active
sites for the binding of protein molecules in a
hydrophilic environment, which may avoid big
conformational changes of the protein structure. This
fact is especially interesting when an enzyme is
immobilized on a surface given the importance seen
in remaining of its conformation structure while is
attached.
The PSb-PFM samples were also characterized
by fluorescence microscopy by using FTSC, which
is a fluorescent molecule with a free amine able to
react with the PFM group on the surface.5 This
technique enables to confirm the PFM reactivity
toward primary amines and shows the homogeneity
of the coating achieved by this method (Figure 3).
After SDS cleaning, the enzyme layer remains
attached, thus a covalent attachment with the coated
surface is confirmed.
To obtain more detailed information about the
enzyme layer formed, the endpoint of the dissipation
shift per frequency shift values (ΔD/Δf) are
represented. It is known that the endpoint ΔD/Δf
value is low in stiffer layers while it increases for an
increasing water content.11 The values corresponding
to the enzyme layer formed by covalent binding
thanks to PFM group is much lower than the
observed in the control (PS+Gln) when the enzyme
is adsorbed nonspecifically to the surface (Figure 5).
Figure 5: ΔD/Δf endpoint of the 1,3-1,4-β-Glucanase formed on a
original PS sensor and on a PS previously modified with PFM by
plasma-grafting technique
Thus, a rigidification of the enzyme layer is
achieved by multipoint covalent immobilization,
which is known to provide the stabilization of the
enzyme.12 As a result, PFM coating by plasma
grafting is presented as an attractive technique to
immobilize enzymes keeping its active points
reactive.
Enzymatic activity. The activity of PSb and PSbPFM incubated with the 1,3-1,4-β-Glucanase in
parallel and were compared to the activity of the free
enzyme. Considering that the immobilization rate
achieved by this technique is 70% (data not shown),
the activity of the covalently attached 1,3-1,4-βGlucanase to the PSb-PFM is around 40% of free
enzyme activity (Figure 6). In addition, these results
show how the enzyme adsorbed to the original PSb
has no activity, suggesting the denaturalization of
the 1,3-1,4-β-Glucanase due to the hydrophobic
nature of this substrate.
Figure 6: Comparation of the enzymatic activity corresponding to the
1,3-1,4-β-Glucanase adsorbed on the original PSb surface, the enzyme
covalently immobilized to the PSb via PFM reaction and the activity of
the free enzyme
4. Conclusions
Modification of polystyrene beads with PFM by
plasma-grafting have been presented as an
interesting methodology for enzyme immobilization.
The enzyme retains 40% of its activity after being
covalently attached on the spherical substrates.
In addition to that, the nature of the surface
modification achieved in this method allows fast and
easy reaction with the molecule of interest thanks to
a click chemistry mechanism, which is provided by
the grafted PFM on the surface. As a result, this
approach has become a versatile tool by enabling the
attachment of a wide range of bioactive molecules to
different substrate geometries.
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