Journal of Membrane Science 213 (2003) 85–95
Simple method for immobilization of bio-macromolecules
onto membranes of different types
Quang Trong Nguyen a,∗ , Zhenghua Ping b , Tuyen Nguyen c , Pierre Rigal c
a
Laboratoire Polymères, Biopolymères, Membranes, UMR 6522, CNRS, Université de Rouen,
Mont-Saint-Aignan Cedex 76821, France
b Department of Macromolecular Science, LMEP, Fudan University, Shanghai 200433, PR China
c Laboratoire d’analyses de Biologie Médicale de l’Yser, 151 bd de l’Yser, Rouen 76000, France
Received 12 August 2002; received in revised form 25 October 2002; accepted 25 October 2002
Abstract
In this method, an intermediate polyelectrolyte layer is first adsorbed on an oppositely charged membrane by electrostatic
interactions. This leads to a charge inversion of the original membrane. Then the bio-macromolecule is bound to the intermediate polyelectrolyte layer, always by charge interactions. The method feasibility was shown with two bio-macromolecules,
glucose oxydase and heparin, which were immobilized on negatively-charged membranes via polyethyleneimine, a cationic
hydrophilic polymer. The immobilization of glucose oxydase on different polymer membranes led to high-activity and stable
membranes for glucose biosensor. The anti-coagulation effect of immobilized heparin was not clearly evidenced in spite of
the effective bio-species immobilization. The good properties of the immobilized enzyme was explained by the hydrophilicity
of the intermediate polyelectrolyte layer, its high density in sites for enzyme binding, and the mild immobilization conditions.
© 2002 Elsevier Science B.V. All rights reserved.
Keywords: Immobilization; Bio-macromolecules; Membranes
1. Introduction
There has been a large number of scientific reports describing various aspects of biofunctional
membranes. Nevertheless, the number of marketed
membranes and devices is still very small in comparison with those for conventional membrane processes.
This is partly due to their high price, since there is
a huge application potential for these membranes in
the fields of medicine and biotechnologies, for direct
clinical applications, for the affinity separation of synthesized biochemicals, or as catalysts in bioreactors or
∗ Corresponding author. Tel.: +33-235147032;
fax: +33-235146704.
E-mail address: [email protected] (Q.T. Nguyen).
in biosensors. Examination of the methods proposed
for the immobilization of bioactive macromolecules
or their fragments reveals that they generally involve
a complex chemistry, either with specially prepared
or fine modification of the support membranes [1,2].
One would expect that a reduction of the manufacture cost of these membranes would boost their
use.
The complex preparation procedure was generally designed to obtain membranes in which the
biofunction of the immobilized species is preserved
for their full activity in applications. For instance, a
site-specific immobilization of biomolecules can be
used to keep the active-site away from the membrane
[3]. However, for the enzymes whose immobilization
via their amine groups does not lead to the active-site
0376-7388/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 6 - 7 3 8 8 ( 0 2 ) 0 0 5 1 5 - X
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Q.T. Nguyen et al. / Journal of Membrane Science 213 (2003) 85–95
blocking, a random covalent immobilization using
these groups to attach the enzymes to a properly
activated support could be favorably envisaged.
Depending on the ability of the starting membrane
(microporous or asymmetric membrane) to be chemically modified, different routes can be used [2,3].
A layer of a polymer with binding sites can also be
deposited on an inert membrane. Two key factors
govern the high efficiency of the bound species: the
concentration of the ready-to-bind groups, and the
hydrophilicity (and the length) of the spacer on the
membrane surface. The former factor directly affects
the content in immobilized biospecies, while the latter
concerns the hydrophilic environment and active-site
accessibility for the biospecies. The spacer and the
binding-group natures as well as the immobilization
methods reported in both scientific journals and in
patents were reviewed by Klein [2]. One can remark,
in examining those methods, that they are generally
quite complex. Moreover, some membranes may undergo morphology change under the effect of the
solvent used in the modification steps.
In this work, we describe a simple method for the
biospecies immobilization onto a variety of support
membranes, provided that the membranes contain a
certain number of surface charges, or can be functionalized into surface charge groups. In a first section
of the paper, the general principle of the method is
described. The method is next illustrated with the
immobilization of two types of biospecies: enzymes
(glucose oxidase and glucoamylase), and heparins.
They are related to two potential applications, namely
sugar biosensors, and hemocompatible membranes.
In a last section, we discuss the advantages and the
drawbacks of the method, and their possible extension
to other membrane-biospecies systems.
2. Principle of the method
It consists of depositing by sorption a layer of a
charged macromolecule from an aqueous solution.
For this, the support membrane must have charges
on its surface. The contact of the membrane with a
solution of a synthetic macromolecule with opposite
charges results in a layer of adsorbed macromolecules
on its surface. The layer of adsorbed macromolecules
serves at the same time as an anchoring material on
the surface and as an active component for the biomacromolecule capture by charge interactions. For the
immobilization, the modified support is put into contact with a solution containing the bio-macromolecule
(a proteins, their fragments, or any bio-active species)
that contain charges opposite to those of the adsorbed
macromolecule layer. This leads to an immobilization
of the proteins by charge interactions, as there are
always both types of charges on protein molecules.
Naturally, if the protein molecule has a total charge
opposite to that of the adsorbed macromolecule, the
quantity of captured species will be greater. Finally, a
chemical crosslinking of the captured species can be
eventually applied for the stabilization of the immobilized layer.
The support membrane needs not to be made of
polyelectrolytes, as a limited number of charges are
required on the membrane (or pore) surfaces. Negative
charges are present on the surface of several commercial membranes, like AN69 hemodialysis membrane (Gambro-Hospal Corp.), sulfonate-containing
polyacrylonitrile-based ultrafiltration membranes
(Rhodia–Orelis), or sulfonated polysulfone (SPSU)based membranes (Nitto Denko, Memtec-PCI, PTA).
Negative charges can also be incorporated onto the
surface of classical membranes like polysulfone
membranes by UV-irradiation of the membrane in
an aqueous solution of an acrylic monomer and a
bis-acrylamide crosslinker [4].
Due to the greater availability of membranes with
negative surface charges, a hydrophilic polymer containing protonated or/and quaternized ammonium
groups are chosen as the polycationic macromolecule for the anchoring to the membrane surface. The polymer can be a commercially available
polyethyleneimine, polyvinylamine, polyallylamine,
polyvinylbenzyl trimethyl ammonium chloride, polyacrylamide with quaternary amine groups, polyvinyl4 pyridine, diethylaminoethyl polysaccharides or
chitosan. Among them, polyethyleneimine is the
most convenient due to their availability and their
well-controlled quality. Some polymers can be easily
crosslinked with bifunctional reagents. Moreover, cocrosslinking of the adsorbed layer with the captured
proteins via their amine groups is possible with the
same bifunctional reagents. The best pH for protein
immobilization on such a modified support membrane
is a pH value higher than the pI value of the protein.
Q.T. Nguyen et al. / Journal of Membrane Science 213 (2003) 85–95
Our method is based on the strong total electrostatic
interactions between two entities bearing a number
of opposite charges. The principle of the method is
reminiscent of that of polyelectrolyte complex membranes. Michaels and Michka [5] used two watersoluble polyelectrolytes of opposite charges, poly
(vinylbenzyltrimethyl ammonium) and poly(styrene
sulfonate), to form ultrafiltration membranes. The
same principle was successfully applied to the preparation of pervaporation membranes for solvent dehydration [6,7]. In both cases, the strong association
of the polyelectrolytes results from both the charge
interactions and a large entropy gain due to the release of the mobile counter-ions from the vicinity of
the polyelectrolyte fixed-charges to the external fluid.
However, as the reaction used in our method involves
charges fixed on the membrane surface (or pore surface), and not soluble macromolecules, the reaction
would not lead to a polyelectrolyte complex of the
same nature as the above-mentioned membranes. Our
systems resemble polyelectrolyte multilayers [8], in
which the charged macromolecules are attached to
the support membrane surface at multiple points.
3. Experimental
87
acrylonitrile in an aqueous medium initiated with a
persulfate–thiosulfate redox system. The content in
negative charges (due to anionic end-groups from the
initiator) was 0.04 equivalent per kg as determined by
ion-exchange capacity measurements. The membrane
was prepared by the wet phase inversion technique.
A 10 wt.% of polyacrylonitrile in dimethylsulfoxyde
was cast on a glass plate with a Gardner knife
and immediately immersed in a bath of 10:90 vol.
DMSO–water mixture for coagulation. The washed
membrane was stored in the wet form.
3.2.2. Preparation of PSU-based asymmetric
ultrafiltration membrane
A sample of sulfonated polysulfone (SPSU) was
first prepared by a reaction of PSU with chlorosulfonic acid in methylene chloride according to a procedure described elsewhere [9]. The content in SPSU
negative charge was 0.8 equivalent per kg as determined by ion-exchange capacity measurements. The
membrane was prepared by the wet phase inversion
technique. A solution of 10 wt.% of PSU and 10 wt.%
of sulfonated PSU in N-methyl pyrrolidone was cast
on a glass plate with a Gardner knife and immediately immersed in a pure water bath for coagulation.
The total ion-exchange capacity of the membrane was
0.4 equivalent per kg.
3.1. Materials
Flat AN69 and Cuprophan hemodialysis membranes were kindly provided by Rhodia and by
Enka Glanzstoff, respectively. Cellulose microporous membrane (0.5 ␮m pore size) was purchased
from Sartorius. Polyethylenimine (PEI) of molecular weight 750,000, polysulfone of Udel P3500 type
(PSU), glucose-oxidase (EC 1.1.3.4, enzyme activity: 218 U/mg), glucose amylase, non-fractionated
heparin extracted from porcine intestinal mucosa
(180 USP/mg) and other chemicals were purchased
from Sigma–Aldrich. Water used for all experiments
was MilliQ-type water (resistivity > 7.5 M).
3.2. Membrane formation and modification
3.2.1. Preparation of asymmetric ultrafiltration
membrane with slightly-charged polyacrylonitrile
A sample of slightly-charged polyacrylonitrile
was first prepared by radical polymerization of
3.2.3. Modification of cellulose membranes by
poly(acrylic acid) grafting
The modification by poly(acrylic acid) grafting
consists first of an immersion of the cellulose membrane in a solution of 0.01 ion g/l of CeIV salt for
15 min at room temperature to form peroxide groups
on the cellulose chains. Next, the washed membrane
was immediately treated with a 0.1 mole/l acrylic acid
solution for 20 h at room temperature for radical polymerization grafting. After washing, the poly(acrylic
acid)-grafted membranes can be stored in the dry
form (for the microfilter), in water or in glycerinated
form (for the Cuprophan membrane).
3.2.4. Fixation of the intermediate PEI layer on
the support membrane and immobilization of the
bio-macromolecule
The adsorption of PEI on membranes was carried
out by dipping the membrane in a 0.5 wt.% PEI solution for ca. 10 min. The PEI solution pH was adjusted
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Q.T. Nguyen et al. / Journal of Membrane Science 213 (2003) 85–95
For the purpose of immobilized-enzyme membrane
comparison, we chose to work with a test solution
of 0.5 m mole/l glucose in pH 5.5 acetate buffer
at 37 ◦ C.
Fig. 1. Experimental set-up used for the study of GOx-immobilized
membrane characteristics: (1) amperometer; (2): Clark-type electrode; (3): thermostated stirred-cell bottom: Clark-type electrode
with membrane and O-ring clip.
by HCl to a value in the range 8.5–10 in order to
obtain a sufficiently protonated form of PEI. After
thorough washing, the membrane was immersed in a
solution of 5 mg/ml of the bio-macromolecules for ca.
30 min at room temperature. The washed membrane
was stored at 4 ◦ C in water.
3.3. Membrane characterization
3.3.1. Activity of glucose oxidase membranes
The glucose concentration in aqueous media was
specifically determined with an amperometric instrument (YSI Clark oxymeter 2510 and sensor) polarized at 0.6 V. Fig. 1 shows the experimental set-up
used for the glucose oxidase (GOx)-membrane study.
The thermostatic measurement cell was well stirred (at
1000 rpm) to minimize the concentration polarization
in the external solution. Glucose is only detected in
the presence of GOx. The prepared GOx membranes
was fastened to the tip of the sensor with an O-ring.
The enzyme catalyzes the conversion of glucose in the
presence of oxygen into hydrogen peroxide. Hydrogen peroxide H2 O2 (electro-active species) was amperometrically determined by measuring the current
resulting from its reduction at the sensor electrode.
The current intensity for a given immobilized-enzyme
membrane can be calibrated with standard solutions of
glucose in 0.1 M acetate buffer at pH 5.5. The glucose
concentration was proportional to the H2 O2 -reduction
current when the current does not exceed 400 nA.
The output current and the saturation level depended
on the nature of the immobilized-enzyme membrane.
3.3.2. Anti-coagulation activity of membranes
with immobilized heparin
The best way to assess the effect of an artificial surface on the blood coagulation would be the determination of the concentration of thrombin-antithrombin III
complex (TAT) in blood [10]. However, this method,
which requires an extracorporal circulation of fresh
blood in contact with the studied surface, cannot be
carried out in our laboratories. We therefore tried the
standard in vitro methods for clotting-problem diagnosis. They consists in measuring the cephaline-kaolin
(CK) time and the prothrombin time (PT).
For all the coagulation tests, blood sample collection on sodium citrate was performed by venipuncture
according to the rules. Human blood with normal
CK time (30 ± 2 s) was prepared for each series of
tests. One milliliter of blood sample was incubated
in silicone-treated test tubes at 25 ◦ C for different times with 4.5 cm2 surface-area samples of the
non-modified membrane, the modified membrane, the
immobilized-heparin membrane, and without membrane, respectively. After centrifugation, the plasma
was mixed with the CK reagent (STA) and was
processed on the “STA compactTM ” instrument, in
which the plasma coagulation was detected by the
damping action on the swinging-ball movement. The
PT was measured on the same instrument, with the
same plasma, after addition of calcium chloride and
thromboplastin (STA). The data were expressed as
the prothrombin index, a ratio of the PT of the normal
blood sample to that of the blood incubated with the
membrane.
3.3.3. Membrane staining
Two different dyes were used: Brilliant Green,
which contains a quaternary ammonium group, and
Ponceau S, which contains four sulfonate groups,
respectively. The latter is water-soluble, while the former is ethanol-soluble. The membrane staining was
performed by dipping the membrane in a 0.1–0.3 wt.%
solution of the dye dissolved in its solvent for ca. 30 s,
then the sample was washed with its solvent until the
washing solvent became colorless.
Q.T. Nguyen et al. / Journal of Membrane Science 213 (2003) 85–95
4. Results
4.1. Response of the electrode with GOx
immobilized on the AN69 membrane
to glucose
Hemodialysis AN69 membrane has sulfonate
groups in its structure due to the polymer material,
which is a acrylonitrile-methallyl sulfonate copolymer. It was directly used for GOx immobilization
without modification.
When the sensor with the enzyme-fixed AN69
membrane was dipped into the 0.5 mM glucose solution, the current signal increases linearly with
time to reach a steady value of 22 nA after ca. 18 s
89
(Fig. 2). The good response signal indicates that the
catalytic activity of the immobilized GOx was high
(Fig. 2), due to an efficient immobilization process,
and an effective preservation of the initial enzyme
on the membrane. The quasi-linear increase in the
electrode current with time in the first part of the
response reflects the transient regime of the transports of the species involved in the glucose oxidation.
The response was rather fast, if one considers that a
12 s-time was required by the sensor to reach a steady
current when a free GOx solution was confined with
a 10 ␮m-thick microporous membrane. Only 6 more
seconds were required for the mass transport through
the 40 ␮m-thick AN69 hemodialysis membrane with
immobilized GOx.
Fig. 2. Responses of the amperometric electrode equipped with different GOx-immobilized membranes when the electrode is dipped in a
0.5 mM glucose solution.
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Table 1
Response time and steady state signal of the amperometric electrode equipped with different GOx-immobilized membranes
Support membrane nature
Symmetric Cuprophan
Symmetric AN69
Asymmetric polyacrylonitrile, UF type
Asymmetric PSU–SPSU blend, UF type
Cellulose microfilter (Sartorius,
0.45 ␮m pore size)
Thickness in
wet state (␮m)
20
40
60
70
110
Treatment
Response
time (s)
CeIV treatment
polymerization
No treatment
No treatment
No treatment
CeIV treatment
polymerization
+ graft
of acrylic acid
+ graft
of acrylic acid
Steady
signal (nA)
12
28
18
20
30
60
22
200
190
170
4.2. Influence of the nature of the support
membrane on the response signal
with glutaraldehyde involves amine groups that are
present in both PEI and GOx molecules.
Different support membranes with the GOx immobilized according to the same procedure as that for
the AN69 membrane were tested with the same sensor and a 0.5 mM glucose solution at pH 5.5 as the
standard test solution. A strong response current was
obtained with each membrane (Fig. 2, Table 1), indicating that GOx was effectively immobilized on the
PEI-treated support membranes, whatever the nature
of the support membrane.
4.4. Bi-enzyme membrane for maltose biosensor
Maltose (or higher oligosaccharides) can be converted to glucose by glucoamylase. In order to determine its concentration, glucoamylase and GOx were
immobilized on the same support membrane. The
glucose produced by maltose hydrolysis is converted
to H2 O2 :
maltose
4.3. Influence of crosslinking on the stability of GOx
immobilized on the AN69 membrane
For continuous glucose monitoring, a glucose
biosensor requires en enzyme membrane with longterm stability in addition to the glucose selectivity and sensitivity in the millimolar range. Enzyme
crosslinking with glutaraldehyde may impart such
a long-term stability to the enzyme by making the
enzyme tertiary structure less sensitive to external
factors.
The GOx-immobilized AN69 membrane was
crosslinked by membrane immersion in a 0.2% glutaraldehyde solution for 10 min. The biosensor with
the crosslinked-GOx membrane was used continuously in a 0.5 mM glucose solution kept at 37 ◦ C.
The glucose solution was renewed every 12 h. The
response current remained constant for 25 days under
continuous monitoring, then decreased rapidly and
irreversibly as a result of the enzyme de-activation.
The efficient stabilization of the fixed enzyme could
be explained by a co-crosslinking of the GOx
molecules with the adsorbed PEI, as a crosslinking
glucoamylase
→
2 glucose
GOx
2 glucose + dissolved O2 → gluconolactone + H2 O2
The two enzymes were co-immobilized on the
asymmetric polyacrylonitrile membrane by simple
immersion of the PEI-treated membrane in a solution
of 2.5 g/l GOx and 5 g/l of glucoamylase. When the
biosensor was put into a 0.5 m mole/l maltose in pH
5.5 buffer, the bi-enzyme membrane gave a steady
current of 90 nA after ca. 60 s, that is the same response time as that with GOx alone on the same
support. This result suggests that the maltose hydrolysis by the immobilized glucoamylase was effective
and was faster than the subsequent glucose oxidation
by GOx. The lower magnitude of the current signal
can be explained by a lower quantity of immobilized
GOx due to the competition, for the same sorption
sites, of the two enzymes from the enzyme mixture.
4.5. Membranes with immobilized heparin
In blood dialysis or filtration with membranes,
heparin is injected in the blood to reduce the risk of
Q.T. Nguyen et al. / Journal of Membrane Science 213 (2003) 85–95
thrombosis induced by the contact of the circulating
blood with the large membrane surface area. The
blood-coagulation activation by an artificial membrane involves very complex processes initiated by
the interactions between blood and the membrane
surface. This activation process can be reduced by
introducing anion-exchange groups on the membrane
surface, or by elaborating membranes from a polymer
blend in such a way that hydrophobic and hydrophilic
microdomains of appropriate size are formed on the
membrane surface [10].
Heparin is an anti-coagulant polysaccharide which
bears negatively-charged groups [11]. In the present
work, we immobilized heparin on the surface of
two PEI-treated membranes, the AN69 and the
PSU–SPSU membranes, and attempted to evidence
the anti-coagulation effect of the fixed heparin. The
used heparin consists of polydisperse chains of molecular weight in the range 2000–30,000 according to
[11]. It should be noted that the negative charges on
the AN69 membrane are thought to cause anaphylactoid reactions within the first 5 min of hemodialysis [12] by generation of a highly active molecule,
bradykinin. The immobilization of heparin on the
membrane could bring positive effect in hemodialysis
due to its better hemo-compatibity.
The heparin immobilization on the membrane was
carried out in a same way as the enzyme immobilization: the membrane with the intermediate PEI layer
was immersed in an aqueous solution of 5 g/l heparin
for ca. 15 min, then thoroughly rinsed with water.
The anti-coagulant activity of the heparinized membranes was first evaluated by measuring the CK time
and the PT time after a contact of the membrane samples with citrated blood for 0.5–1.5 h. No effect of the
blood-membrane contact was observed for all samples (CK time: 31 ± 2 s, PT index: 100%, same values as for the reference blood). However, when the
heparinized membrane samples were left in contact
with citrated blood for 3 days at 20 ◦ C, the measured
CK time (38–45 s), and the PT index were lengthened (PT index: 41–67%). Nevertheless, we are careful
not to come to the conclusion of an anti-coagulation
effect, as the used procedure was non-conventional
due to the long contact time required for the blood
sample.
The absence of a clear anti-coagulation effect of
the immobilized heparin in CK and PT tests may
91
indicate either a non-adequate procedure for the
anti-coagulation effect estimation, a non-effective
heparin immobilization, or a de-activation of heparin
after immobilization. The streaming-potential measurements did not give interpretable data, probably
because of the complex structure of the GOx membranes (with swellable charge layer and complex
spatial charge distribution). We tried a qualitative
detection technique, the specific staining of the membrane at different steps of the immobilization procedure. The staining technique based on organic dyes
containing ionic groups is widely used in biology but
rarely in the membrane science. As the immobilization technique involves changes in surface charge,
anionic (Red Ponceau S) and cationic dyes (Brilliant
Green) were used for the staining.
Fig. 3 shows the colors obtained with the AN69
stained with the two dyes at different steps. The original AN69 was well stained in green by the cationic
dye (Fig. 3, top-left), but practically not stained by
the anionic dye (not shown). The membrane treated
with the PEI polycation was more colored by the red
anionic dye (Fig. 3, top-right). After the adsorption of
the anionic heparin, the membrane was again strongly
stained by the green cationic dye (Fig. 3, bottom-left).
The contact of the latter membrane with an aqueous
solution of the red anionic dye show a deeper dyeing,
Fig. 3. Colors obtained with the AN69 membrane stained with
cationic “Brilliant Green” and anionic “Ponceau Red” dyes at different steps of the heparin-immobilization procedure. Staining of:
the initial membrane by the cationic dye (top-left); the membrane
treated with the PEI polycation by the red anionic dye (top-right);
the membrane after adsorption of the anionic heparin by the green
cationic dye (bottom-left); after contact of the latter membrane
with an aqueous solution of the red anionic dye (bottom-right).
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Q.T. Nguyen et al. / Journal of Membrane Science 213 (2003) 85–95
Fig. 4. Colors obtained with the PSU–SPSU blend membrane
stained with cationic “Brilliant Green” and anionic “Ponceau Red”
dyes at different steps of the heparin-immobilization procedure.
Staining of: the initial membrane by the cationic dye (top-right);
the membrane treated with the PEI polycation by the red anionic
dye (top-left); the membrane after adsorption of the anionic heparin by the green cationic dye (bottom-left); after contact of the
latter membrane with an aqueous solution of the red anionic dye
(bottom-right).
indicating a slight staining by the red dye (Fig. 3,
bottom-right).
The asymmetric PSU–SPSU membranes in different adsorption steps showed similar behaviors in the
staining experiments (Fig. 4). The more intense staining of the asymmetric PSU–SPSU membranes (Fig. 4,
left side) compared with the symmetric ones (AN69,
Fig. 3 top-right and bottom-left) after PEI treatment and heparin adsorption, respectively, indicates
larger amounts of dyes adsorbed on the asymmetric
Fig. 5. Close examination of the heparin-immobilized PSU–SPSU
membrane after successive staining with “Brilliant Green” and
“Ponceau Red” dyes.
membranes than on the symmetric ones. Such results
are consistent with the large internal surface area for
the polyelectrolyte adsorption (Fig. 4, left), while
only the external faces of the AN69 are accessible to
the polyelectrolytes.
When a heparinized asymmetric membrane which
is first stained by the green cationic dye (Fig. 4,
bottom-left) is treated with a red anionic dye solution
in a second step, some red-colored zones are visible
underneath the green-color layer (Fig. 4, bottom-right
and Fig. 5). It appears clearly that the red anionic
dye penetrated through the heparin layer to stain the
adsorbed PEI layer underneath (Fig. 5).
5. Discussion
The different values of the response time and the
steady current obtained with the GOx-immobilized
membranes (Table 1) reflect the change in the H2 O2
transport rate towards the electrode and the rate of
H2 O2 production from glucose by the GOx-catalyzed
reaction. The transport rate of a solute through a membrane generally depends on the membrane structure
and morphology. As there was no pressure gradient
through the membrane, the solute molecules were
transported towards the electrode by diffusion under
the concentration gradients induced by the consumption of H2 O2 and glucose at the electrode surface
and in the GOx membrane, respectively. We expected
that the more porous (or swollen) the membrane, the
larger the amount of the analyte penetrated into the
membrane, and the higher the transport rate.
Although the used support membranes have quite
different morphologies, we observed that the thinner
the membrane, the shorter the response time (Table 1).
This means that the transport rate depends more on
the membrane thickness, i.e. the species permeability
did not vary much with the support membrane nature.
It may appear surprising that the gel-type membranes
like Cuprophan and AN69 hemodialysis membranes
did not exhibit a much larger resistance to glucose
transport in comparison with the microporous membranes. Since hemodialysis membranes are designed
to let small molecules like glucose penetrate freely,
the diffusivity of glucose in the membrane water
phase would be not much smaller than that in water.
If one takes into account the ca. 100% total swelling
Q.T. Nguyen et al. / Journal of Membrane Science 213 (2003) 85–95
of these membranes in water, a partition coefficient of
ca. 0.5 would be obtained under the condition of no
penetrant exclusion from the absorbed water phase in
the membranes. Thus, the hemodialysis membranes
show an overall penetrant distribution coefficient in
their gel-structure comparable to that in the cellulose microfilter, which has ca. 50% porosity. The
similar magnitude of the permeability coefficient in
different membranes explains the dominant role of
the membrane total thickness in the response time.
The results also indicate that the layers of adsorbed
macromolecules did not add any significant transport
resistance to that of the support membranes.
The observed sequence for the response signal magnitude is (Table 1): asymmetric polyacrylonitrile ∼
=
asymmetric PSU–SPSU blend > cellulose microfilter
> AN69 membrane > Cuprophan membrane. The
magnitude of the steady response signal reflects the
concentration of H2 O2 produced in the immobilizedenzyme membrane. The rate of H2 O2 production
from glucose by the GOx-catalyzed reaction can be
described by the Michaelis–Menten equation:
v=
k[GOx]0 [glucose]
Km + [glucose]
As the same immobilization procedure was used for
all membranes and the support membrane is not in
direct contact with the immobilized enzyme, we can
reasonably assume that the Michaelis constant Km and
the rate constant k are independent of the nature of the
support membrane. With these assumptions, the rate of
H2 O2 production is proportional to the quantity of (immobilized) enzyme, as the glucose concentration was
fixed in the experiments. The results (Table 1) indicate
that the quantities of immobilized GOx for the asymmetric polyacrylonitrile, the asymmetric PSU–SPSU
blend and the cellulose microfilter were seven and
eight times higher than that for the AN69 membrane,
which was itself 40% higher than that for the Cuprophan membrane. The much higher quantity of immobilized GOx for the asymmetric and the microporous
membranes can be explained by the high internal area
onto which were adsorbed the intermediate macromolecule and the enzyme, which cannot penetrate into
the hemodialysis membranes due to size exclusion.
All the above results are consistent with a two-step
kinetics (oxidation of glucose into H2 O2 followed by
93
diffusion of H2 O2 to the amperometric electrode) in
which the H2 O2 diffusion is the rate-limiting step.
Concerning the heparin immobilization, the membrane staining results point to a change in the charge
of the membrane to that of the polyelectrolyte used in
the sorption step, as the captured dye was always the
dye whose molecule contains groups with charges opposite to those of the polyelectrolyte. We infer that the
polyelectrolyte was effectively immobilized onto the
membrane. The absence of clear anti-coagulant activity could be due to either an unsuitable in vitro coagulation tests, or the lack of anti-coagulant activity of the
immobilized heparin. In fact, the heparin immobilized
on the hemodialysis AN69 membrane did show an
anti-coagulant activity in vivo (in sheeps and humans),
as recently reported Thomas in a Symposium [13].
The successful immobilization of either heparin or
enzymes on PEI-treated membranes of different morphological structures suggests that the method based
on polyelectrolyte multilayers could be used for the
immobilization of various bio-macromolecules on
different support membranes. Although this method
involves multiple-charge interactions as does the
formation of polyelectrolyte complex membranes,
the resulting materials have different structures and
physico-chemical properties. Contrary to the case of
polyelectrolyte complexes, where infusible materials
with intimately-mixed polyelectrolytes of mutuallyneutralized charges are obtained whatever the starting
charge ratio of the polyelectrolytes [5], an adsorbed
polyelectrolyte layer whose total charge exceeds that
of the surface results from the contact of the membrane with an oppositely charged polyelectrolyte in
solution [14,15]. In other words, the membrane surface charge is reversed after the adsorption of the
oppositely charged polyelectrolyte.
The versatility of the method can be explained
by its principle, i.e. the formation of polyelectrolyte
multilayers. A recent paper showed effectively that
the polyelectrolyte multilayers are very stable [14].
The charge inversion phenomenon was theoretically
studied by several groups in the recent years. Joanny
et al. [15] predicted that there should be an overcompensation of the surface charge by the polyelectrolyte, which leads to the charge inversion, whatever
the adsorption conditions. Our experiments of specific staining by charged dyes confirm the charge
inversion at each step of polyelectrolyte sorption from
94
Q.T. Nguyen et al. / Journal of Membrane Science 213 (2003) 85–95
Fig. 6. Schematic representation of the mechanism of enzyme and heparin immobilization. Adsorption of the PEI intermediate layer and
immobilization of enzyme (top-right) and heparin (bottom-right).
low ionic strength media. However, theories showed
that the polyelectrolyte multilayers can be effectively
formed at high ionic strength. They also show that
highly-charged supports are not required. This seems
to be true in our work: the PAN asymmetric membrane which has only 0.04 equivalent per kg anionic
groups (end-groups from the initiator) gave a GOximmobilized membrane as good as the support membranes with higher negative charges. For amphoteric
bio-molecules like proteins, a good adsorption on PEItreated membranes is expected when the proteins are
at pH higher than their isoelectric point in solutions.
From the point of view of the final membrane
sorption properties, the high-activity of the enzyme
membrane can be explained by the high content in
binding sites (cationic groups) on the intermediate
PEI layer. The adsorbed polyelectrolyte layers do not
completely screen out the dye molecules that bear
the same charge. In other words, the mesh size of the
adsorbed layers is large enough compared with the
electrostatic screening distance of the polyelectrolyte
charges. This situation is understandable if we admit
that parts of the chain collapsed on the surface by
charge interactions, and parts stretched towards the
bulk liquid medium as tails and loose loops. Such an
accessibility to the species from the external solution
would favor the reactions/interactions that involve the
immobilized bio-macromolecules.
On the basis of the above data, a scheme representing the mechanism of enzyme or heparin immobilization is proposed (Fig. 6). The results concerning
the GOx membranes illustrate the numerous advantages of the method: high binding capacity due to
the large number of charge-binding sites on the PEI
layer, high compatibility of the anchoring polymer
with hydrophilic bio-molecules, simple and mild immobilization procedure (absence of toxic chemicals)
. . . . We also expect a shielding of the proteins or biomacromolecules from the hydrophobic interactions
of the membrane surface by the PEI layer. Due to
these advantages, membranes with high bio-activity
and high stability of bio-macromolecules could be
manufactured at a reasonable cost.
Another interesting feature is that the support membrane can be chosen a priori to fit optimally the target
application. The original membrane morphology is
normally preserved in the immobilization procedure,
as the latter does not require any membrane drying or
contact with organic solvents. One can therefore adjust the transport resistance of the support membrane
as well as their selectivity by selecting its thickness,
its pore size (e.g. by using ultrafiltration, nanofiltration, reverse osmosis, or dialysis membranes) or its
physico-chemical properties (e.g. hydrophobicity). A
specific organization of the bioactive species in the
membrane system can be eventually envisaged (e.g.
Q.T. Nguyen et al. / Journal of Membrane Science 213 (2003) 85–95
immobilization of two bio-macromolecules specifically on the two membrane faces, immobilization of
different bio-macromolecules on layered membranes).
For instance, a selective screening of macromolecules
which can reach the enzyme layer can be obtained by
immobilizing the enzyme on one side of an ultrafiltration layer. The range of applications or fundamental
studies can thus be considerably extended.
6. Conclusion
The simple method proposed for immobilization
bio-macromolecules on membranes seems to be versatile, in that different active species can be easily
immobilized on membranes of diverse natures. The
versatility of the method is probably due to its principle, i.e. the formation of polyelectrolyte multilayers.
The preparation of immobilized-enzyme membranes
for catalytic conversion, or membranes with immobilized active fragments for affinity separation would
be attractive applications of this simple method, as
high contents in active species on industrial-type
membranes (including membranes in ready-to-use
modules like hollow fiber modules) could be obtained
at low manufacture cost.
Acknowledgements
The authors acknowledge the Minister of Education
of China for the Funding of the stay of Q.T.N. at
Fudan University in the frame of the program of Key
Laboratory of Polymer Molecular Engineering.
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