Download Atomic Layer Deposition on Biological Macromolecules: Metal Oxide

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LETTERS
Atomic Layer Deposition on Biological
Macromolecules: Metal Oxide Coating
of Tobacco Mosaic Virus and Ferritin
2006
Vol. 6, No. 6
1172-1177
Mato Knez,*,† Anan Kadri,‡ Christina Wege,‡ Ulrich G o1 sele,† Holger Jeske,‡ and
Kornelius Nielsch†
Max-Planck-Institute of Mikrostructure Physics, Weinberg 2, D-06120 Halle, Germany,
and Department of Molecular Biology and Plant Virology, UniVersity of Stuttgart,
Pfaffenwaldring 57, D-70550 Stuttgart, Germany
Received February 22, 2006; Revised Manuscript Received April 18, 2006
ABSTRACT
Decoration of nanoparticles, in particular biomolecules, gathered high attention in recent years.1-7 Of special interest is the potential use of
biomolecules as templates for the fabrication of semiconducting or metallic nanostructures.1-7,26 In this work we show the application of
atomic layer deposition, a gas-phase thin film deposition process, to biological macromolecules, which are frequently used as templates in
nanoscale science, and the possibility to fabricate metal oxide nanotubes and thin films with embedded biomolecules.1-13
Since the development of atomic layer deposition (ALD) in
the 1970s14 the process has mainly been used in the
microelectronics industry and related research areas. In this
rapidly growing research area most developments are devoted
to process optimization for technologically relevant materials
such as electroluminescent films or high-k materials.15-19
During the ALD process the target samples are exposed to
a precursor molecule from the gas phase allowing the
precursor to build a layer (in the ideal case a monolayer) on
the substrate. Subsequent purging of excess precursor gas
and exposure to a second precursor lead to a reaction on the
substrate, building a layer of the target material.16,18,19,28,29
This cycle can be repeated until the desired layer thickness
is obtained. In most cases the deposition requires a comparatively high temperature (200-500 °C) and therefore
cannot be applied to temperature-sensitive bioorganic materials. However, recently deposition of aluminum oxide at room
temperature onto polyethylene was accomplished.20 In the
same manner atomic layer deposition can be applied to some
biological macromolecules, e.g., the tobacco mosaic virus
(TMV) and ferritin.
TMV has already shown the ability to act as template for
the growth of semiconductor and metal particles and
wires.5,7,21 The virus is composed of about 2130 identical
proteins which are helically arranged around a single-strand
RNA, forming a 300 nm long tubular structure with an outer
* To whom correspondence may be addressed. Tel.: +49-345-5582929. Fax: +49-345-55-11223. E-mail: [email protected].
† Max-Planck-Institute of Mikrostructure Physics.
‡ Department of Molecular Biology and Plant Virology, University of
Stuttgart.
10.1021/nl060413j CCC: $33.50
Published on Web 04/27/2006
© 2006 American Chemical Society
diameter of 18 nm and a hollow channel with a diameter of
4 nm.22-25 The viruses can attract each other, either headto-tail, forming a linear aggregation of viruses, or side-byside, leading to two-dimensional (2D) or three-dimensional
(3D) structures if they are protonated or deprotonated, i.e.,
by changing the pH value of the medium.11,26 The details of
this aggregation process have, however, not yet been
explored in detail. TMV is a highly defined nanostructure
and because of its extraordinary stability as compared to other
biomolecules is a very interesting molecule for nanotechnology. It can resist temperatures of up to 80 °C without
destruction of its integral shape, it can be handled in a pH
range of around 2.8-8.0 fairly long (minutes to hours), and
it can be dried building a crystal-like structure without
destruction of the single virions, which is of major importance for our research.25,27 Apart from the TMV some of the
most frequently used biological template molecules in
nanotechnology are ferritin molecules, a spherical protein
complex with an iron oxide core, which can be adsorbed to
surfaces forming a hexagonal arrangement.13
The experimental procedure of the ALD deposition on the
biological macromolecules is schematically shown in Figure
1. First, a suspension of the biomolecules is dried on a solid
support. Subsequently the sample is transferred to the ALD
chamber. In the next step the molecules are exposed to a
precursor from the gas phase, which in our case is either
titanium tetraisopropyl oxide (TIP) or tetramethylaluminum
(TMA) (see methods). The excess of the precursor gas is
removed by purging while a layer of precursor molecules
remains chemisorbed or physisorbed on the substrate. In the
Figure 1. Overview of the experimental process for the coating of the biomolecules by ALD. After drying on a substrate, the biomolecules
are transferred to the ALD chamber and exposed to TMA or TIP vapor, the excess of the precursor in the gas phase is purged and the
substrate is successively exposed to water vapor. The reaction products and the excess of water are purged. After 20-100 cycles the coated
biomolecules are obtained. After removal from the ALD chamber they can be resuspended and analyzed by TEM.
next step the substrate is exposed to a second precursor,
which is in our case water, hydrolyzing the adsorbed first
layer to the desired metal oxide. However, biomolecules
often contain accessible amines and alcohols and bound water
molecules which are not removed by drying. Water, in
particular, can instantaneously react with TIP or TMA to
build the first metal oxide layer on the biomolecules. The
precursor layers in the successive cycles are hydrolyzed with
the second precursor. After the hydrolysis reaction, the excess
water molecules and the reaction products are removed by
purging, to prevent side reactions during the next cycle. The
metal oxide layer on the substrate is, after formation of the
nucleation layer, growing linearly with each cycle.
For our process suspensions of TMV, prepared as described elsewhere,7 and ferritin (from horse spleen, SigmaAldrich), were dried on a silicon wafer or on a piece of
laboratory film (Parafilm) in an incubator at 35 °C until no
liquid could be observed. A large part of the ALD reaction
chamber surface was covered with hydrophobic laboratory
film (Parafilm) to decrease the amount of water bound to
the chamber wall and the purging time after the water pulse,
otherwise the purging time would need to be significantly
higher.20 The predried sample was transferred into an ALD
chamber (Savannah, Cambridge Nanotechnology Inc.) and
evacuated at 1 × 10-1 Torr at 35 °C for 2 h in order to
remove remaining loosely bound water from the pellets. After
the drying procedure, the deposition process (see schematic
Figure 1) was started and run for 20 (for TMV) to 100 (for
ferritin) cycles at 2 × 10-1 Torr at 35 °C. In our experiments
we used TMA (ABCR GmbH) or TIP (Sigma-Aldrich) as
first precursor and water as second precursor in order to
deposit Al2O3 or TiO2, respectively. Pulsing times were 1.0
s for TIP, 0.2 s for TMA, and 1.3 s for water. Exposition
times of 20 s and purging times of 60 s were used for all
pulses. Purging was done with Ar gas with a flow rate of 10
sccm. After the ALD process, the biomolecules were
Nano Lett., Vol. 6, No. 6, 2006
resuspended in water and a portion was treated in an
ultrasound bath for 5-10 min. A 10 µL droplet of each
suspension was deposited onto a carbon-coated holey
transmission electron microscope grid (400 mesh, Plano).
After 10 min the excess liquid was removed with filter paper
and the sample examined in a transmission electron microscope at 200 kV (Philips CM20 twin equipped with a LaB6
cathode). The obtained a growth rate was 0.5-0.9 Å per
cycle for Al2O3 and TiO2.
Figures 2 and 3 show dispersed TMV on a carbon-coated
TEM grid. In parts c and d of Figure 2, the TMV was coated
with TiO2 from TIP/H2O. Analogous results were obtained
after deposition of Al2O3 from TMA/H2O (see Figures S1
and S2 in Supporting Information). The dark area around
the outer viral surface and in the internal cavity in parts c
and d of Figure 2 clearly shows an enhanced contrast as
compared to the untreated viruses (Figure 2 a,b)). None of
the viruses are stained by any means. The strong enhancement of the contrast of the biomolecules clearly comes from
the oxide film with higher density than the viral protein. The
coverage of the viruses is complete, i.e., all of the viruses
treated by ALD have an enhanced contrast. However, the
coverage of the outer surface is, apart from the contact areas
of the TMV with the substrate and with neighboring TMV,
conformal. By ultrasonication after resuspension of the TMV,
the TiO2 and Al2O3 can partly be removed from the outer
surface of the TMV, and viruses with surface-covered interior
channel only can be obtained (see Figure 2d).
A deposition of metals or metal oxides in narrow channels
can only be obtained if the channel surface is accessible to
the precursor. However, the precursor in the gas phase has
a certain spatial extension and kinetic energy, which makes
it unlikely that narrow tubes can be entirely filled. If a critical
channel diameter is reached, the transport of the precursor
molecules into the channel is blocked and the structure
remains hollow, but the accessible end of the structure will
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Figure 2. Upper images: TEM (200 kV) image of untreated TMV. The Y-shaped gray area originates from the holey carbon film on the
TEM grid. In this area some darker shadows (on image b marked with white lines) can be seen. These shadows come from untreated TMV
adsorbed on the TEM grid. Image a is the same image without markers. Image c: TEM (200 kV) image of TMV treated with TiO2 by
ALD. The coverage of the viruses with TiO2 reaches 100%. The viruses are embedded in an amorphous TiO2 film visible as a blurry area
interconnecting the viruses. Image d: After ultrasonication the TiO2 is partially removed from the outer surface and mainly the inner
channel of the TMV remains covered with TiO2 (visible as dark lines).
be clogged. The TMV has such a narrow channel and it is
expected that the inner channel will not be completely filled
with the metal oxide. Due to the natural assembly intermediates of TMV, or cleavage while treating mechanically (by,
e.g., ultrasonication or resuspension by shaking), some
fragments of the virus (e.g., 20S disks24b) occur. Such
fragments, if they are short enough, align perpendicular to
the substrate and can be observed by TEM in cross-section
view.
After the ALD process we observed such TMV fragments
which in several cases show a hollow metal oxide tube inside
the hollow viral channel (see Figure 3). On a single TEM
grid several hundreds of such virus fragments can be seen
(Figure 2c is representative). Among them at least 40%
showed a pore inside the metal oxide covered inner channel.
The diameter of the inner viral channel is 4 nm and so
is the outer diameter of the TiO2 tube or Al2O3 tube inside
the biotemplate. However, the pore inside the TiO2 tube
has a diameter of 1-1.5 nm (see Figure 3), and with Al2O3
even smaller diameters can be reached (see Supporting
Information), which puts these tubes among the smallest
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metal oxide nanotubes which could be observed so far. The
fragments that do not show pores inside the TiO2 are assumed
to originate from the end part of the virus. With Al2O3 the
pore is much smaller (see Supporting Information, Figure
S2), presumably because the precursor molecule (TMA) is
much smaller than TIP and thus can cover the surface of
even smaller channels. The resulting pores are at the
resolution limit of the TEM used for those samples. Common
for all observed particles is that they are usually embedded
in a thin amorphous metal oxide film, unless the interconnecting film is removed, e.g., by ultrasonication. Upon
exposure to the electron beam in high magnification, the
smooth interconnecting thin metal oxide film is instantaneously affected, i.e., only for a very short period (1-2 s)
does the film appear homogeneous. After 1-2 s, bright
irregularly distributed spots in the interconnecting metal
oxide film appear, which show the sensitivity of the thin
film to high-energy irradiation. However, the holes inside
the viruses can be seen also before the destruction of the
thin film, which shows that they are not produced by the
electron beam.
Nano Lett., Vol. 6, No. 6, 2006
Figure 3. Image a: TEM (200 kV) image of TMV treated with TiO2 by ALD without successive ultrasonication. A disk from destroyed
TMV (circular particle) embedded in an amorphous TiO2 film can be seen in cross section view. The TiO2 covering the interior channel
appears to be hollow with a pore diameter of 1-1.5 nm with a wall thickness of 1 nm. The covered inner channel of the viruses appears
brighter along the axis, confirming the assumption of a hollow TiO2 nanotube. Image b: Magnification of a further TiO2-covered TMV
disk showing a hollow area inside the TiO2-covered interior channel of the virus. Image c: Recolored image b. The orange circle represents
the viral protein sheath. The blue color shows the TiO2 coating of viral surface (outer surface and channel surface). The surrounding gray
area is the embedding amorphous TiO2 film. Image d: Sketch of a cross section of a TiO2 covered TMV with the same colors as in image
c. In the top part of the virus no pore is visible in the center. This part represents the assumed clogged area of the inner viral channel. Image
e: Magnification of a further TiO2-covered TMV disk showing a clogged interior channel of the virus. Image f: Recolored image e.
Recently30 carbon nanotubes were decorated with tungsten
and Al2O3 on their outer surface by ALD. The resulting
nanotubes have a very similar diameter. Although those
nanotubes have a larger length than ours, the deposited Al2O3
nanotubes cannot easily be released from the substrate, since
the carbon nanotube is comparatively stable. Biomolecules,
however, can easily be decomposed by a number of
procedures, e.g., UV light, higher temperatures, acids or
bases, or enzymes. For our experiments, however, it is
reasonable to keep the biomolecular shell for stability
reasons, since the Al2O3 and TiO2 nanotubes with their very
thin tube walls are expected to be very fragile. Another very
important advantage of biomolecules is the large variation
of chemical and physical properties that can be controlled.
Amino acids can be hydrophilic or hydrophobic, polar or
unpolar, or, e.g., charged. Those properties can influence the
chemical behavior of the molecule. In the case of TMV (e.g.,
the vulgare strain) the accessible outer molecular surface of
the virus is dominated by hydrophilic amino acids (ser, thr,
arg, asp) of which a large part is charged (arg, asp). In
addition some water molecules are tigthly bond to the
proteins. Hydrophobic amino acids (trp, val) can be accessed;
however, they are predominantly buried in a hydrophilic
surrounding (see Figure 4). The surface of the viral channel
shows a similar chemistry, however with different amino
acids. In particular for ALD reactions with water (TMA, TIP,
etc.) no selectivity on the molecular surface with naturally
occurring TMV can be expected. The distances that need to
be bridged by the metal oxide to overgrow the hydrophobic
parts is in the range of a few angstroms. Assuming that the
oxide layer grows from both sides, assisted by the firmly
bound water molecules, the hydrophobic areas should be
Nano Lett., Vol. 6, No. 6, 2006
Figure 4. Model of a TMV disk (vulgare strain) generated with
data from refs 22, 23, 24a, and 33. The amino acids are colored
white for hydrophobic amino acids, blue for water molecules, green
for polar (hydrophilic) amino acids, and red for charged (hydrophilic) amino acids. On both surfaces, the outer viral surface and
the inner channel surface, no spatially extended hydrophobic areas
are found.
overgrown within the first two to five ALD cycles. The
selectivity of deposition, however, can be altered either by
chosing molecules with large hydrophobic surface areas or
by genetically designing such molecules. In those cases the
oxide layer growth (in particular with water-sensitive reactants) in these areas is expected to be very slow or zero, if
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from the described depositions of Al2O3 and TiO2, we expect
that similar reactions at low temperatures can be accomplished with other oxides such as ZrO2 or ZnO and even
nitrides such as TiN. If low-temperature deposition of metals
became possible (e.g., with proper precursors), ALD on
biological or organic material could become a very important
technique for the fabrication of flexible electronics.
Figure 5. TEM (200 kV) images of ferritin molecules treated with
Al2O3 (image a) and TiO2 (image b) by ALD. The images show
ferritin molecules embedded in an amorphous free-standing Al2O3
and TiO2 films. The darker gray areas originate from the holey
carbon film on the TEM grid. The black spots in image b show the
iron oxide core of ferritin. The films have cracks, and on image b
the free-standing film is rolled up from the side, which was caused
by the electron beam from the TEM.
not only a few cycles are performed and the hydrophobic
areas are overgrown from neighboring hydrophilic sites. The
proof for this thesis, however, needs additional experiments
which will be performed soon.
We applied ALD to deposit TiO2 or Al2O3 also onto
ferritin and obtained a free-standing metal oxide film with
embedded ferritin molecules (see Figure 5). The TEM images
show a compact film with embedded ferritin molecules (dark
dots show the ferritin core consisting of iron oxide). The
contrast of the oxide can only be observed if the film is
preserved. Upon ultrasonication the interconnecting metal
oxide film is removed and the ferritin molecules appear
untreated. This effect is expected, since ferritin offers no
cavity for the precursor to penetrate and thus the metal oxide
can only be attached to the outer surface and thus easily
mechanically removed. Nevertheless, this method offers a
possibility to interconnect ordered films of ferritin molecules
as they were shown by Yamashita.13 Embedding of protein
assemblies in macroscopic films may not be new,31,32 but
the application of ALD at low temperatures allows for precise
control of the thickness of the matrix films even in the
subangstrom range and is thus of high interest for bioinspired
nanoscale structuring. In addition embedding of biomolecules
not only becomes possible in organic or polymer films but
also can be performed with inorganic compounds.
The described results show the potential of ALD for the
decoration of biological macromolecules. On one hand, as
can be seen from the decoration experiments of TMV, narrow
pores and channels with less than 4 nm in diameter can be
accessed by the precursors and nanotubes can be fabricated,
on the other hand the metal oxide deposition of the ferritin
molecules shows that ALD can easily be expanded to the
fabrication of thin films of interconnected biomolecules and
other temperature-sensitive nanoparticles. Preliminary results
show that the presented method of ALD coverage of
biological macromolecules can be extended to DNA as well.
The chemical or physical properties of the biomolecules can
be altered with the metal oxide coating. Even the replication
of highly complex shapes of biomolecules which cannot be
generated in a laboratory otherwise can be performed. Apart
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Acknowledgment. Three of the authors (M. Knez, U.
Gösele, and K. Nielsch) appreciate the financial support by
the German Ministry of Science and Education, BMBF, via
Research Contract FKZ 03N8701. The authors H. Jeske, A.
Kadri, and Ch. Wege are grateful to the Landesstiftung
Baden-Württemberg for funding within the Kompetenznetz
“Funktionelle Nanostrukturen”, project C5. We thank Dr.
Hesse (MPI-Halle) for support in the TEM investigations.
Supporting Information Available: Images of TMV
after deposition of Al2O3 by ALD and TiO2- and Al2O3covered TMV disks. This material is available free of charge
via the Internet at http://pubs.acs.org.
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