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Hybrid graphite lmcarbon nanotube platform

for enzyme immobilization and protection


A. Kondyurin
a
, I. Levchenko
b,a
, Z.-J. Han
b,a
, S. Yick
a,b
, A. Mai-Prochnow
b
J. Fang
b,c
, K. Ostrikov
b,a,
*
, M.M.M. Bilek
a
a
School of Physics, The University of Sydney, Sydney, NSW 2006, Australia
b
Plasma Nanoscience, CSIRO Materials Science and Engineering, P.O. Box 218, Lindeld, NSW 2070, Australia
c
School of Physics, University of Melbourne, Parkville, VIC 3010, Australia
A R T I C L E I N F O
Article history:
Received 24 May 2013
Accepted 18 August 2013
Available online 27 August 2013
A B S T R A C T
A novel platform consisting of a multilayered substrate, activated graphite-like carbon lm,
and dense forest of long, vertically-aligned multiwall carbon nanotubes grown by the
chemical vapor deposition is designed, fabricated, and tested for covalent immobilization
of enzymatic biocatalysts with the aim of protecting them from shear forces and microbial
attacks present in bioreactors. The covalent bonding ensures enzyme retention in a ow,
while the dense nanotube forest may serve as a protection of the enzymes from microbial
attack without impeding the ow of reactants and products. This platform was demon-
strated for the two reference enzymes, horseradish peroxidase and catalase, which were
immobilized without degrading their biological activity. This combination of an activated
carbon layer for an efcient immobilization of biocatalysts with a protective layer of inert
carbon nanotubes could dramatically improve the efciency and longevity of enzymatic
bio-catalysis employed in a large variety of advanced biotechnological processes.
Crown Copyright 2013 Published by Elsevier Ltd. All rights reserved.
1. Introduction
Surface-supported carbon nanostructures such as nanotubes
and vertically-aligned graphene akes are fascinating candi-
dates for numerous applications in pharmaceutical, agro-
chemical, and healthcare product fabrication [1,2]. Unique
physico-chemical, mechanical and optical properties of the
carbon nanostructures allow for the efcient adsorption,
retention, and trapping of various reagents on the surface
[3]. The processes that utilize enzyme biocatalysts [4,5] are
of a critical importance for the above applications. However,
a very high cost of enzymes is a major drawback of the tech-
nique. Signicant productivity increases would follow from
longer-term use of expensive enzymes that commonly suffer
from shear forces and microbial attacks in continuous
reagent ows [6,7]. Covalent immobilization of the enzymes
onto a scaffold structure allows them to be stably held in
the ow and can also improve their long-term stability under
hostile process conditions [8,9]. It also provides an easy way
to recover and recycle enzymes from the reaction mixture
and to control the product concentration, keeping it below
the poisoning threshold, by continuous removal of product.
Continuous reagent ows and microbial attacks are major
issues that severely damage the enzyme catalysts and signif-
icantly reduce their lifespan. One of the attractive approaches
to protect the enzymes against these attacks in a uid-bed
reactor consists in encapsulating them in nanoporous sub-
strates [10,11]. The pores in the substrates must be engi-
neered carefully so that they are small enough to exclude
microorganisms, whilst being sufciently transparent and
connected to provide efcient transportation of reactants
from the reaction mixture [12]. Besides, many materials used
for the fabrication of nanoporous substrates are often reac-
tive. These limitations motivate the search for other possibil-
0008-6223/$ - see front matter Crown Copyright 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbon.2013.08.028
* Corresponding author.
E-mail address: Kostya.Ostrikov@csiro.au (K. Ostrikov).
C A R B O N 6 5 ( 2 0 1 3 ) 2 8 7 2 9 5
Avai l abl e at www. sci encedi r ect . com
ScienceDirect
j our nal homepage: www. el sevi er . com/ l ocat e/ car bon
ities for simultaneously immobilizing and protecting en-
zymes for bio-catalytic processes.
Here we report on designing and testing a novel hybrid
platform for the immobilization and protection of the enzy-
matic biocatalysts from shear forces and microbial attacks.
The platform is based on a specially designed silicon
(Si)/silicon oxide (SiO
2
)/iron (Fe) substrate covered with an
activated graphite-like carbon lm used to covalently attach
the biocatalyst, and a dense protective forest of vertically-
aligned multiwall carbon nanotubes (CNT) grown on the
graphite-like carbon lm. We have fabricated this platform
and demonstrated effective covalent immobilization of the
two reference enzymes, horseradish peroxidase (HRP) and
catalase, on the activated carbon underneath the nanotubes.
We have also demonstrated that covalently immobilized en-
zyme preserve their activity after immobilization in this plat-
form. The mechanism of immobilization is by means of
highly-reactive surface-embedded radicals that form covalent
bonds with macromolecules in solution. The protective CNTs
are inactive, do not immobilize protein molecules and do not
interact with the enzymes except to screen them.
2. Experimental
2.1. Nanotube synthesizes
The CNTs are synthesized on a solid substrate from a carbon
rich gaseous phase. In this process, the carbon-containing
gaseous precursors (e.g., methane CH
4
, ethylene C
2
H
4
etc.)
rstly dissociate into atomic or molecular carbon species on
the surface of catalyst nanoparticles [13], and then the nucle-
ation occurs as these carbon species diffuse into the catalyst
nanoparticles, reach a supersaturated state, and then segre-
gate from the surface of nanoparticles to form a nanotube
cap. Subsequently, the growth of nanotubes is sustained by
the continuous incorporation of carbon atoms via bulk and/
or surface diffusion [1416].
The prepared substrates (100 nm of SiO
2
on a 0.5-mm Si
wafer) with 1 nm of Fe (which catalyses the growth of carbon
nanotubes in carbon-reach atmosphere) were loaded into a
quartz tube of a thermal furnace (MTI, OTF-1200). Then, the
temperature in the furnace was ramped to 750 C in a mixture
of hydrogen (H
2
) and acetylene (C
2
H
2
) (900/100 sccm) gases at
atmospheric pressure. Under these conditions, thin metal (Fe)
layer fragments to nano particles which serve as catalytic
centers for the nanotube growth. After a certain growth time
(typically 10 min), the supply of H
2
and C
2
H
2
was terminated,
and samples were cooled down to room temperature under a
continuous argon (Ar) ow. The growth parameters were se-
lected to ensure fast growth of the dense carbon nanotube
pattern simultaneously with the formation of carbonous
underlayer of 510 nm in thickness. Schematic of the experi-
mental setup used for growing nanotube arrays is shown in
Fig. S1, Supporting Information. SEM images of the typical ar-
ray are shown in Fig. 1.
The process of CNT synthesis results in the deposition a
carbon layer on the substrate surface as well as the growth
of the nanotubes. Therefore, the nal structure of the
substrate consists of ne CNTs standing on the top of a
carbon under layer. In our work, nanotubes with a small
number of walls, about 5, and with a very low level of intrinsic
defects were grown on catalyst-coated silicon substrates. This
allowed us to selectively characterize the proteins attached to
the CNTs and to the graphite-like lm on the substrate.
2.2. Sample preparation
The samples for characterization of the enzyme attachment
were prepared in a two-step process. In the rst stage, carbon
nanotube arrays were grown on silicon substrates measuring
1 1 cm. In the second stage, the arrays were incubated in a
buffer solution of horseradish peroxidase (50 lg/ml HRP con-
centration in PBS buffer, pH 5.5, incubated overnight, then
washed 6 times in PBS, with the last wash in mQ water) as
well as in catalase (200 lg/ml catalase concentration in PBS
buffer, pH 5.5, incubated overnight, then washed 6 times in
PBS, with the last wash in mQ water). The samples for the
FTIR, XPS and SEM characterization were allowed to dry over-
night prior to analysis. After analysis, samples were washed
in sodium dodecyl sulphate (SDS), which is a detergent capa-
ble of disrupting physical interactions and fully unfolding
proteins (SDS 2% water solution, 70 C, 1 h). In the experi-
ments with blocking agent, the samples were incubated for
3 h in 10% ethanol solution of (2,2,6,6-tetramethylpiperidin-1-yl)
oxyl (TEMPO) free radical trapper. After washing in ethanol,
the sample was incubated in HRP solution as described above.
The activity was tested using test reagent on the as-prepared
(wet) samples.
2.3. Characterization and microanalysis
Attenuated total reection (ATR)Fourier transform infrared
(FTIR) spectra were recorded at an incidence angle of 45
using a Digilab FTS7000 FTIR spectrometer tted with an
ATR accessory (Harrick) and a trapezium germanium crystal.
Generation of spectra with sufciently high spectral band
resolution and signal-to-noise ratio required 500 scans at a
resolution of 1 cm
1
. The surface of the samples was dried
using dry air ow before data collection. The effect of surface
treatment was characterized by the difference in spectra ob-
tained before and after the treatment. All spectral analyses
were made using GRAMS software. The elemental composi-
tion of the sample surfaces was characterized using an
X-ray photoelectron spectroscopy (XPS) spectrometer
(Specs-XPS, mode XP-50 High Performance Twin Anode with
Focus 500 Ellipsoidal Crystal Monochromator and PHOIBOS
150 MCD-9 analyzer). Binding energies of photoelectrons
(within a range of 501200 eV) emitted from the samples irra-
diated with K
a
X-rays produced by a dual anode were used to
measure concentrations of the elements. Sufcient number
of scans and slit width was maintained to get high signal/
noise ratio. Areas of C
1s
, O
1s
and N
1s
element peaks were cal-
culated using CASA XPS and divided by relative sensitivity
factors for each element. The concentration of each element
was calculated as an atomic percentage. Atomic force micros-
copy (AFM) images of samples before and after incubation in
enzyme solution were collected on a PicoSPM instrument in
tapping mode at a scan rate of 0.427 lines/s over an area of
1 1 lm. Analysis of the AFM images was performed using
the WSxM software (version 3, Nanotec Electronica S.L., Spain
288 C A R B O N 6 5 ( 2 0 1 3 ) 2 8 7 2 9 5
[17]). SEM images were made using a Zeiss Ultra Plus, AURIGA
model scanning electron microscope (FE-SEM). Transmission
electron microscopy (TEM) characterization was done by a
JEOL 3000F high-resolution transmission electron microscope.
Raman spectra were recorded using confocal micro-Raman
spectroscopy (inVia Renishaw, 514 nm laser source).
3. Results and discussion
3.1. Structure of the nanotube arrays
Fig. 1(a) illustrates the structure of the proposed platformcon-
sisting of multilayered Si/SiO
2
/Fe substrate, activated carbon
layer, and dense forest of multiwall carbon nanotubes.
Rendered images of 3D models of the two used reference en-
zymes, catalase and horseradish peroxidase, are shown in
Fig. 1(b) and (c), respectively. Fig. 1(d) and (e) shows the low-
and high-resolution scanning electron microscopy (SEM)
images of the carbon nanotube arrays on the substrate. The
low- and high-resolution TEM images of the as-prepared
nanotubes, collected from the array and transferred to the
microscopy grid, are shown in Fig. 1(f) and (g). From this im-
age one can see that the arrays exhibit a very high density
of vertically-aligned nanotubes with a mean length reaching
20 lm. The diameter of the nanotubes reaches 20 nm, with
the number of walls of about 1020.
Top view of the CNT array on the substrate, and side view
of CNT array detached from the substrate are shown in
Fig. 2(a) and (b). SEM images of the CNT arrays after incuba-
tion in enzyme (catalase) solution and washing in SDS are
shown in Fig. 2(c) and (d). Fig. 2(e) shows FTIR ATR spectra ta-
ken from the samples incubated in catalase solution. The
lines at 1650 and 1540 cm
1
are attributed to the Amide I
and Amide II vibrations of amide bond in the enzyme back
bone. A spectrum from plasma immersion ion implantation
treated ultra-high-molecular-weight polyethylene (UHMWPE)
with a monolayer of immobilized catalase [18] is shown for
comparison (see Supporting Information for more details
about the plasma immersion ion implantation treatment).
Spectra taken from a silicon wafer and the reference
CNT sample after incubation in a buffer solution without
proteins were subtracted. The presence of the amide lines
indicates that catalase is present. The XPS spectra of CNTarray,
CNT array with attached HRP, and CNT array after HRP attach-
ment and washing in SDS detergent are shown in Fig. 2(f).
3.2. Chemical composition and structure of the samples
X-ray photoelectron spectroscopy was used to study the
chemical composition of the samples prior to and after the
incubation in horseradish peroxidase solution and sodium
dodecyl sulphate detergent washing cycles, see Figs. 2(f) and
3. The strong C1s peak at 285 eV
originates from the carbon nanotubes. The XPS spectrum
of the CNT substrate shows an O1s oxygen peak attributed
to a slight oxidation of the graphite-like carbon layer and does
not show a nitrogen peak. The shoulders of the carbon C1s
peak at 287 and 288 eV, corresponding to carbon in CO and
C@O groups of the protein molecules, appear only after incu-
bation in HRP enzyme solution. The strong nitrogen peak at
Fig. 1 (a) Schematic of the hybrid graphite lmcarbon nanotube platform. Enzymes are immobilized on a layer of activated
carbon beneath the carbon nanotubes. (b, c) Catalase and horseradish peroxidase used for testing the platform. (d, e) Lowand
high resolution SEM image of the carbon nanotube arrays on the substrate. (f, g) Low-and high-resolution TEM images of the
as-grown carbon nanotubes. A colour version of this gure can be viewed online.
C A R B O N 6 5 ( 2 0 1 3 ) 2 8 7 2 9 5 289
400 eVand oxygen peak at 532 eV that also appear after incu-
bation in HRP solution are attributed to oxygen and nitrogen
atoms in protein molecules. After washing of samples in SDS,
these peaks remain in the spectra indicating that a signicant
fraction of the enzyme is covalently attached.
Fig. 4(a) shows the nitrogen and oxygen compositions of
the samples calculated from the XPS spectra. The concentra-
tion of oxygen in the CNT is about twice the detection level.
The concentration of nitrogen is zero. After HRP attachment,
the surface concentration of oxygen and nitrogen increases to
5.5% and 3.7%. After washing in SDS detergent, the surface
concentration of oxygen decreases to 4.8% and the concentra-
tion of nitrogen decreases to 2.5%. The concentration of oxy-
gen can be inuenced by oxidation of the substrate during
manipulations, while the concentration of nitrogen is associ-
ated only with the amount of the protein immobilized [19].
Changes in the nitrogen concentration indicate that about
33% of the HRP protein is removed during the SDS detergent
wash while 67% remain strongly bonded to the material.
Fig. 4(b) shows an AFM image of catalase macromolecules ad-
sorbed on a smooth polystyrene lm. Their size agrees well
with that observed in the high resolution SEM characteriza-
tion of the CNT samples.
We have assumed that the covalent bonding of proteins in
this platform is based on free radical reaction. To prove this,
we have blocked free radicals in CNT samples with a free
radical trapper agent. The intact CNT samples were treated
by (2,2,6,6-tetramethylpiperidin-1-yl)oxyl and then incubated
and washed; the amount of removed protein in the detergent
wash increases to 88% while only 18% now being strongly
bonded on the samples. We believe that the bonding mecha-
nism is the same as that described in [20]. The residual bond-
ing that occurs after application of the trapping agent is
expected due to the continuous emergence of radicals.
The plasma treatment of the unprotected carbon-covered
surfaces creates radicals in the embedded in the surface
which provide efcient covalent bonding of proteins. Speci-
cally, on untreated surfaces 100% of the adsorbed proteins are
washed away by the method described [20]. In contrast, plas-
ma treated surfaces typically retained over 50% of the ad-
sorbed protein layer indicating that these were covalently
bonded [20]. In the platform reported here, about 67% of the
adsorbed layer was covalently bonded and this dropped to
18% of the adsorbed layer when the surface was exposed to
a radical trapping agent prior to incubation with the protein.
This shows that the covalent bonding of proteins occurs due
to a radical reaction resulting in efcient enzyme immobiliza-
tion on this platform as described in [20].
3.3. Enzyme immobilization
SEM images of the nanotubes with immobilized catalase en-
zymes are shown in Fig. 5. The catalase molecules appear
as small spheres with a diameter of about 20 nm, correspond-
ing to the size of catalase macromolecules [21,22]. To obtain
an image of the bottom of the substrate, the nanotubes were
Fig. 2 SEM, FTIRandXPScharacterizationof the carbonnanotube arraybefore andafter incubationinenzyme solution. (a) Top
view of an as-prepared CNTarray. (b) Side view of CNTarray detached from the substrate. (c, d) Top view of the array after
incubationinenzyme (catalase) solutionat low(c) and high(d) magnication. (e) Amide I andAmide II lines inFTIRATRspectra
of catalase on the CNTarray and ultra-high molecular-weight polyethylene (for comparison). The spectra of the silicon wafer
and spectra of a control CNT sample after incubation in buffer without protein are subtracted. (f) XPS spectra of pristine CNT
array (line 1), CNTarray with attached HRP (line 3), and CNTarray after HRP attachment and washing in SDS detergent (line 2).
The edge of the p-plasmon (which is a characteristic of electron excitation in regular graphitic structures like CNTs) is marked
with arrows on these lines. The edge for the sample with attached HRP shifted to lower energies (line 3, 367 eV), and then
shifted back to 380 eV for the sample washed in SDS (line 2). A colour version of this gure can be viewed online.
290 C A R B O N 6 5 ( 2 0 1 3 ) 2 8 7 2 9 5
removed mechanically. SEM images were taken from above
looking down at the surface and from the edge of the cracked
substrate. A large number of catalase molecules can be seen
on the carbon coated surface of the substrate. We conclude
from these images that most of the protein molecules are at-
tached to the carbon lm under the nanotube forest and not
to the CNTs themselves.
Raman spectra of the as prepared (before immersion in the
enzyme solution) CNT samples show D and G peaks attrib-
uted to the CNTs and the carbon lm underneath (Fig. 6).
The peaks have a mixture of broad and narrow components.
For analysis, the Raman spectra were tted with 4 Gaussian
functions. The two narrow peaks at 1328 cm
1
(D-peak) and
1602 cm
1
(G-peak) are attributed to vibrational modes in
the graphene structures in the CNTwalls, and the two broad
peaks at 1327 cm
1
(D-peak) and 1577 cm
1
(G-peak) are
attributed to carbon in the lm underneath. All of the
samples give a strong G peak which evidences the high-
quality structure of the carbon nanotubes. The inuence of
incubation in enzyme (HRP) solution and subsequent SDS
washing on the Raman spectra can be observed as a change
in the D/G intensity ratio. This ratio is 1.1 for the pristine
CNT samples (typical for the samples consisting of the dense
pattern of carbon nanotube and a thin carbonous layer under-
neath [23]) and increases to 1.64 after immobilization of the
enzyme molecules. It is then partially restored to 1.44 after
washing the samples with SDS. This indicates that (i) proteins
adsorbed on the nanotube surface disturb the vibrations of
the CNTs; (ii) a considerable fraction of the enzymes adsorbed
on the CNTs is removed from the samples by SDS washing
(and hence, was attached non-covalently).
A signature of the radial breathing-like mode (RBM) was
also found in the Raman spectra (Fig. 6). We recall here that
the arrays comprise multi-walled nanotubes of 10 nm dia.,
thus consisting of a few atomic carbon layers. The RBM peak
is quite strong for the as-grown nanotubes, indicating that
Fig. 3 High-resolution XPS spectra of CNT array (black),
CNTarray with attached HRP (blue), and CNTarray after HRP
attachment and washing in SDS detergent (red). The C1s
peak at 285 eV originates from carbon nanotubes. The
shoulders at 287 and 288 eV, corresponding to carbon C1s in
CO, CN, and C@O groups in the protein, appear after HRP
incubation. The O1s peak at 532 eV, originating fromoxygen
in the protein, appears after incubation in HRP. The N1s
peak at 400 eV corresponds to nitrogen in protein. No
nitrogen signal was found on CNT arrays before protein
attachment. (A colour version of this gure can be viewed
online.)
Fig. 4 (a) Elemental content of the CNT arrays before and
after exposure to enzyme solution. Very weak oxygen and
nitrogen peaks can be noticed before protein attachment.
After protein attachment, strong O1s and N1s signals
appear. After washing with detergent, the intensity of the
O1s and N1s peaks slightly decreases, but remains strong.
(b) AFM image and the associated line prole taken from a
polystyrene lm with adsorbed catalase macromolecules.
The size of the catalase macromolecule as observed here
corresponds to the size of the features found on the bottom
of the CNTarray samples. A colour version of this gure can
be viewed online.
C A R B O N 6 5 ( 2 0 1 3 ) 2 8 7 2 9 5 291
the nanotubes have only a few walls. The RBM peaks
disappear after incubation in enzyme solution, indicating an
increased degree of structural disorder in the CNTs.
Another important characteristic of the structural order of
carbon nanotubes is the p-plasmon shoulder of the XPS C1s
line (Fig. 2). In the spectrum of the pristine CNT sample (line
1, Fig. 2f), the p-plasmon shoulder extends to 376 eV. In the
spectrum obtained from the nanotube sample with the
attached HRP (i.e., after enzyme incubation, line 3 in Fig. 2f),
the p-plasmon shoulder shifted to lower energies and ends
Fig. 5 SEM images of catalase protein on carbon nanotubes and substrate. (a) and (b) show the top of the CNT forest, (c) and
(d) show the surface underneath, viewed at an edge near the cracked substrate (d) and on the at surface after removal of the
CNTs (c).
Fig. 6 Raman spectra and schematic of the enzyme attachment. (ad) Raman spectra of the nanotube array before and after
incubation in enzyme (HRP) solution, and after subsequent SDS washing. The D/G = 1.1 peak ratio is the lowest for the as-
prepared nanotube array (a), the highest (1.64) after incubation in enzyme solution (b), and somewhat restored (1.44) after
SDS washing (c). The radial breathing-like mode peak at 189 cm
1
is relatively strong for the as-prepared CNT array but
signicantly weakened after incubation and SDS washing (d). A colour version of this gure can be viewed online.
292 C A R B O N 6 5 ( 2 0 1 3 ) 2 8 7 2 9 5
at 367 eV. The spectrum taken after washing the sample in
SDS (line 2) has the shoulder at 380 eV, i.e., it shifted back
close to the value for the pristine sample (line 1, 376 eV).
Thus, the Raman and XPS spectrometry and SEM images
have shown that the enzymes are physically adsorbed on
CNTs, and covalently immobilized on the carbon lm beneath
the CNTs. The enzymes are weakly (physically) adsorbed on
the CNTwalls by van der Waals forces and are easily removed
by washing since the energy of physical interaction is very
weak, not exceeding fractions of eV. In contrast, the chemical
bonds established between the enzymes and the carbonous
underlayer are much stronger and cannot be destroyed by
SDS [19,20].
For applications in biotechnology, the biological activity of
the enzymes immobilized on the activated carbon layer be-
neath the nanotube pattern must be maintained. Previous
experiments have demonstrated that the enzymes immobi-
lized on similar plasma activated carbon thin lms remain
biologically active [24,25]. In comparson with enzymes in
solution, an activity of 50% was observed which is as ex-
pected for undegraded enzymes given the reduced diffusion
of reactants and products due geometrical differences alone
[19].
3.4. Mechanical stability and protective capabilities of the
hybrid platform
To investigate the mechanical stability of the hybrid plat-
form, we placed the enzyme-treated samples in 24 well
plates on a shaker (Benchtop Excella E1 shaker, New Bruns-
wick Scientic) set to 30 rpm rotation with an amplitude of
2 cm. The plates were lled with LB solution (Peptone 10 g;
yeast extract 5 g, and NaCl 10 g per liter of water), so the
samples were completely immersed in the liquid. The sam-
ples were tested in this regime for 12 h. After that, to exam-
ine the sample structure, we dried them by the critical point
drying (CPD) procedure (note that the carbon nanotube sam-
ples usually collapse during drying in open air). The samples
were immersed in 100% ethanol, then transferred to the CPD
chamber (BAL-TEC CPD030 Critical Point Dryer) and dried
using liquid CO
2
for 3 h at the critical point (+31.1 C, 1000
PSI). After that, SEM was used to examine the structure of
Fig. 7 SEM images of the upper surface of sample after 12 h in the shaker and critical point drying (a) and after drying in
open air (b). No changes in the structure of surface after critical point drying were found as compared with the as-prepared
sample (Fig. 2a). (c) Schematic of the enzyme protection against microbiological attack. Enzymes are attached on the carbon
lm deposited onto the substrate surface and protected by a dense and long carbon nanotube forest, while bacteria cannot
reach the protected enzyme molecules. (d) and (e) Low and high resolution SEM images of the B.subtilis on the surface of the
nanotube pattern after 12 h of incubation. Bacteria were trapped in the upper layer of the nanotube pattern. A colour version
of this gure can be viewed online.
C A R B O N 6 5 ( 2 0 1 3 ) 2 8 7 2 9 5 293
the CNT pattern. It was found that the pattern was not chan-
ged. A SEM image of the top surface of the CNT pattern after
12-h treatment in the shaker is shown in Fig. 7(a). The SEM
image of the collapsed sample after drying in open air is
shown in Fig. 7(b).
To check the protective capabilities of the platform
against microbiological attack, we have conducted a direct
experiment on the interaction of live bacteria (Bacillus
subtilis, B.subtilis) with our platform. For this, we have
incubated the samples with a live culture of B.subtilis for
12 h and then made SEM images after critical point drying
(see the relevant description above). A schematic diagram
of the immobilized enzyme protection against microbiolog-
ical attack is shown in Fig. 7(c). This experiment has dem-
onstrated that the bacteria cannot penetrate into the
protective CNT forest, and are trapped on its surface, as
seen in Fig. 7(d) and (e) (see the low-resolution SEM image
of B.subtilis on the nanotube pattern in the Supporting
Information provided).
We propose that this structure could be useful as a support
for the immobilization of enzymes for continuous ow enzy-
matic processing [2628]. The gaps between carbon nano-
tubes are about the same size as the diameter of the
nanotubes (1020 nm), allowing dissolved reactants to reach
the carbon layer beneath where most of the enzymes are
immobilized whilst blocking bacteria whose typical size is
2002000 nm. The height of the CNTs is about 10 lm, much
higher than the enzyme molecules (1020 nm) so damaging
microbes cannot approach the enzymes. The tall densely
packed CNT forest will also protect enzyme molecules against
the hydrodynamic pressure caused by uid ow, if the en-
zyme-rich substrate is used in continuous high ow biochem-
ical reactors.
4. Conclusions
In this work, a novel platform for the covalent immobiliza-
tion and protection of the biocatalysts was designed, fabri-
cated and tested. The reference enzymes (horseradish
peroxidase and catalase) were immobilized on an activated
graphite-like carbon lm, deposited onto the multilayered
substrate and covered by densely packed, long multiwall
carbon nanotubes. The large (and relatively unchanged
by SDS washing) protein signatures observed by FTIR ATR
and XPS spectroscopy indicate that the majority of the en-
zyme molecules are covalently attached on graphite-like carbon
lm beneath the nanotubes. On the other hand, most of the
enzyme was removed from the walls of nanotubes by
washing in SDS detergent, as evidenced by the partial
recovery of the Raman and XPS signals associated with
the regular CNT structure. Thus we conclude that the pro-
posed platform efciently adsorbs and covalently immobi-
lizes enzymes beneath the protective nanotube forest, thus
protecting them from the shear forces and microbial at-
tacks present in the bioreactors. In further work, we plan
to test the developed system for efciency of protection
in microbial environments typical for the industrial appli-
cations. Also, we will consider the possibility to use other
nanostructures, such as ber bundles and carbon nano-
cones, as a protective part of our platform.
Acknowledgements
This work was partially supported by CSIRO and the Austra-
lian Research Council.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.carbon.
2013.08.028.
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