Making Electrical Contact to Single Molecules
by
Wolfgang Fritzsche1*, Konrad J. Böhm2, Eberhard
Unger2, J. Michael Köhler1
1Microsystems Department, Institute of Physical High Technology,
P.O. Box 100 239, 07702 Jena, Germany
2Research Group Molecular Cytology / Electron Microscopy,
Institute of Molecular Biotechnology,
P. O. Box 100 813, 07708 Jena,
Germany
*corresponding author: w.fritzsche@b3.ipht-jena.de ,
FAX 0049-3641-657744
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This is a draft paper
for a talk at the
Fifth
Foresight Conference on Molecular Nanotechnology.
The final version has been submitted
for publication in the special Conference issue of Nanotechnology.
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Abstract
We are interested in the characterization of electron transport phenomenon
through single (bio)molecules. A prerequisite for electrical measurements
is the contacting of molecules in the nanometer range. The presented paper
describes various steps toward this goal, starting from statistically
distributed molecules (namely, microtubules) adsorbed on a microstructured
surface. Monitored by scanning force microscopy and video-enhanced
microscopy, adsorption parameters were studied and optimized, including
protein-mediated adsorption, induced orientation, and controlled
desorption. Line structures connecting the adsorbed molecules to
prestructured electrodes were created by means of electron beam-induced
deposition in a scanning electron microscope.
Introduction
The characterization of the electron transport through single molecules is
of great interest for a variety of fields, reaching from microelectronics
to biology. Methods for single molecule detection and manipulation have
been established, e.g., based on scanning probe techniques or optical
tweezers. An interesting property of molecules is the electron transport
mechanism. First studies demonstrated the possibility of contacting and
subsequent electrical measurement of single molecules with nanometer
dimensions (Bockrath et al., 1997; Dai et al., 1996). Approaches in this
direction are usually based on scanning probe techniques, due to the random
distribution of the molecules over the surface. These techniques are used
for electrical measurements (which are hampered by the complex behavior of
the tip-sample contact), and/or for detection of the molecules. The
detection of the molecules in the scanning force microscope (SFM) is
followed by the structuring of electrodes (e.g., using electron beam
lithography). Concluding, the basic scheme of a single molecule measurement
consists of four steps (Fig. 1): In a first step, the molecules of interest
are adsorbed onto the isolating substrate, usually from a liquid phase
(Fig. 1a). The substrate is often already equipped with some electrical
contacts. The second step includes the visualization of the molecule
distribution, mostly by SFM (Fig. 1b). In a third step the molecule is
contacted, either through the conductive scanning tip or by the structuring
of electrodes (Fig. 1c). If a structuring step is involved, the images from
SFM are usually used for localization of the molecules of interest.
Finally, the fourth step includes the electrical characterization (e.g.,
current-voltage, Fig. 1d).
This paper presents methodological developments which are mainly focused on
the first (adsorption) and thethird (contacting) step of the
above-mentioned scheme. It describes experiments with microtubules as model
molecules. Microtubules (MTs) are protein assemblies with essential
functions in cell architecture and cellular transport, with dimensions in
the micrometer (length) and nanometer (width) range. The adsorption
behavior of MTs onto microstructured electrode surfaces was optimized, and
contact structures between the electrodes and the biomolecules were created
using electron beam-induced deposition.
Fig. 1: Scheme of a typical experiment for electrical
characterization of a single molecule. a) Adsorption of molecules on
prestructured substrates. b) Visualization of the adsorbed molecules,
usually by SFM. c) Structuring of connective structures between electrodes
and molecules, using electron beam-based methods. d) Electrical
characterization.
Materials and Methods
Substrates
Arrays of electrode pairs were structured from ~100 nm gold (primed with a
~5 nm titanium layer) on a thermally oxidized silicon wafer according to
standard photolithographic techniques. The pairs had contact pads of
400x200 µm (for convenient contacting by macroscopic electrodes) connected
to electrodes of 1 µm width, which were separated by a 12 µm gap (cf. Fig.
4).
For adsorption experiments test structures of gold lines on oxidized
silicon wafers with comparable structure sizes were used.
Microtubules
Microtubules (MTs) were prepared as described previously (Vater et al.,
1995). Microtubules assembled from tubulin both with and without
microtubule-associated proteins (MAP2s, (Vater et al., 1986)) were studied.
MTs were purified from brain by three cycles of temperature-dependent
disasssembly/reassembly as described (Shelanski et al., 1973; Vater et al.,
1983). Using phosphocellulose column chromatography (Weingarten et al.,
1976), the microtubule-associated proteins (MAPs) were separated from the
tubulin, which represents the main protein of MTs. The MAP2 was obtained
from the pool of MAPs by elution from phosphocellulose with 1 M NaCl,
followed by heat treatment and gelfiltration. In the presented study, MTs
were assembled from tubulin both with and without MAPs and stabilized by 20
mM taxol.
For adsorption experiments, the assemblies were applied to a substrate of
microstructured gold lines on oxidized silicon.
Two different methods of application were used to study the orientation of
the MTs: The first method ("running droplet") included the repeated
application of droplets on the tilted (~45°) chip, so that the droplets
were running over and leaving the surface without staying. The other method
("staying droplet") applied a droplet on a laying chip, followed by an
incubation for 5 minutes. In both cases, the chips were then washed in
buffer and aqueous taxol-solution followed by air-drying.
The influence of MAPs on the adsorption properties of MTs was investigated
using video-enhanced microscopy. For this purpose, a solution of MAP2 was
filled into a glass flow chamber (about 20 mm length, 2 mm width, 0.2 mm
height). After binding of MAP2 to the glass and washing, a region of the
surface was imaged and a suspension of MAP-free MTs was injected under
simultaneously microscopic control. The reversibility of MT binding was
checked by washing the chamber in taxol-containing buffer with high ionic
strength (0.5 M NaCl), breaking of MAP-tubulin binding (Vallee, 1983).
For contacting, MTs with MAPs were adsorbed to the microstructured gold
electrodes described above.
Microscopy
Scanning electron images of uncoated samples were obtained by a digitized
scanning electron microscope (SEM) DSM 960 (Zeiss). For deposition of
electron beam-induced deposited (EBD) lines the slow scan axis of the SEM
was disabled for usually 20 minutes.
For scanning force microscopy a NanoScope III (Digital Instruments (DI),
Santa Barbara, CA) with a Dimension 3000 or Multimode head (with lateral
scan ranges of~130 µm) was used. The microscopes were operated in the
tapping mode using tapping mode etched silicon tips (DI).
MT adsorption onto glass was studied by video-enhanced differential
interference contrast microscopy, using a microscope Axiophot (Zeiss)
equipped with the image processing system Argus 50 (Hamamatsu). Image
processing was performed following instructions in (Allen et al., 1981).
The directions of adsorbed MTs were determined from scanning force
micrographs after import in the program NIH Image 1.61 (developed at the
U.S. National Institutes of Health and available on the Internet at
http://rsb.info.nih.gov/nih-image/). The fast scan direction (left to
right) is thereby assigned as 0°, from which the measured angles opened
counterclockwise.
Results
MT adsorption onto microstructured substrates
The adsorption of MTs both with and without MAPs onto microstructured
substrates was studied using SFM. Therefore, substrates with gold and
silicon oxide surfaces were incubated with assembly solutions, prior to
washing and air-drying. The SFM investigation revealed that no adsorption
takes place in the case of MTs without MAPs. Only after addition of MAP2 to
the assembly solution the samples exhibited rod-like structures of some
micrometer length, which is the typical appearance of MTs (cf. Figs. 3, 5).
Height measurements yielded 7-10 nm, but were hampered by a surface
roughness of up to 3 nm.
MT substrate binding by pre-adsorption of MAP2
The mechanism of substrate binding of MT-MAP2 complexes was investigated by
subsequent incubating the surfaces in MAP solution and washing, prior to
addition of MTs. SFM images of gold and silicon oxide surfaces with MAP2
revealed an increased surface roughness due to the occurrence of globular
structures with diameters of ~25 nm (measured as height) in a high density
(up to 15 per µm2). The addition of MTs to such surfaces resulted in
MT-adsorption, comparable to samples of MT-MAP2 complexes. Again, the
sample surface was contaminated by the described globular structures.
Fig. 2: Reversible attachment of microtubules formed from pure
tubulin to MAP2-covered glass surfaces monitored by video-enhanced DIC
microscopy. All images show the same area of the glass surface (cf. the
three distinct features on the surface, marked by arrowheads). Scale bars =
10 µm. a) Image of the glass surface after binding of MAP2. b) Image after
inflow of the microtubule suspension and binding of microtubules to MAP2.
c) Image of the glass surface after release of the microtubules from the
MAP2-covered surface, which was caused by the inflow of buffer with high
ionic strength (0.5 M NaCl).
Reversible attachment of MTs
The reversible attachment of microtubules formed from pure tubulin to
MAP2-covered glass surfaces was investigated by video-enhanced microscopy.
Glass slides incubated with MAP2 revealed no particular structure
comparable to the results from SFM of MAP2-coated surfaces. A region with
three distinct features (marked by arrowheads in Fig. 2) was chosen for
further observation. After inflow of the microtubule suspension adsorbed
microtubules became visible (Fig. 2b). Incubation of the adsorbed MTs with
buffer of high ionic strength resulted in MT removal (Fig. 2c).
Preferred MT orientation by flow adsorption
To increase the yield of MTs positioned parallel to the electrodes a flow
adsorption method was used. Thereby, droplets of assembly solution (MTs
with MAP2) were repeatedly applied to the tilted substrates (inset, Fig.
3a). They moved along the substrate surface in parts of a second, and left
it afterwards. For comparison, droplets were also applied onto substrates
which were laying flat, and incubated there for some minutes (inset, Fig.
3b). Samples prepared according to both methods were then washed and
air-dried prior to SFM imaging.
Both procedures yielded samples covered with MTs (Fig. 3). For characterization of the MT-orientation the directions of all MTs (as given by the long axis)
were determined. In the case of flow adsorption, the frequency distribution
revealed a clear peak at about 90°, which agrees with the flow direction
(from bottom to top, plot in Fig. 3a). The effect of flow is clearly
visible in the comparison to the histogram of the control sample, which
exhibits a random distribution covering the whole range nearly equally
(plot in Fig. 3b). Experiments with different flow directions of the
washing fluids and with a variation of the direction of the receding
meniscus yielded no differences in the observed orientation, thereby
confirming the crucial role of the first adsorption for the MT orientation.
Fig. 3: The influence of the liquid dynamic onto the adsorption
process. Microtubule-MAP complexes were applied in flow (from top to
bottom, a) or as a sitting droplet (b). The orientation of the adsorbed
microtubules was determined from the SFM images and is presented as
histogram (bottom). The peak in the distribution for the flow-adsorbed
proteins clearly demonstrates the induced orientation compared to the
random behavior of the droplet-adsorbed microtubules.
Electron beam-induced deposition of electrode structures The
creation of lines by electron beam-induced deposition (EBD) was tested on
the microstructured substrates. Therefore, the slow scan direction of the
SEM was disabled. This forces the electron beam to cycle along one scan
line, which results in the build-up of a line structure. Some test
structures created on silicon oxide between gold electrodes are shown in
Fig. 4a: Three intersecting lines create a connection through the electrode
gap. A typical feature of the EBD lines produced in our SEM is a
cone-shaped feature at the beginning of the scan line, with a height
clearly exceeding the line height. This effect is probably due to a
prolonged duration of the beam in this point compared to the rest of the
line, and will be subject of further studies. The growth rate of the lines
is significantly increased on conducting surfaces, e.g., on gold-sputtered
surfaces (Fig. 4b). The use of EBD lines for contacting molecules is
demonstrated on a rather macroscopic dust particle situated nearby the
electrodes (Fig. 4c): An EBD line connect each electrode with the molecule
(Fig. 4d).
Fig. 4: Nanometer structures by electron beam induced deposition
(EBD). Scanning electron micrographs. Scale bars are 3 µm (a,b) and 10 µm
(c,d), respectively. a) Microstructured gold electrodes (broad structures
at the left and right) on oxidized silicon were connected by writing three
EBD lines. Note the low contrast of the image due to the isolating
background. b) EBD line written and visualized on a conductive background
(ca. 10 nm gold coating). c,d) A dust particle, located nearby the
electrode gap, is used as a test molecule for demonstrating the contacting
by a EBD line (d).
Contacting of MTs in electrode gaps
For demonstration of the contacting principle, MT-MAP2 complexes were
adsorbed in (or nearby) electrode gaps and visualized by SFM (Fig. 5a). A
long MT intersecting one of the electrodes was chosen for further
experiments (MT in Fig. 5a). The sample was then transferred to the SEM,
and an EBD line was written bridging the gold electrode (E) with the free
end of the MT. Fig. 5b shows the sample in the SEM, the EBD line is faintly
visible (arrows).
Fig. 5: Contacting of a single microtubule. a) MTs adsorbed in
an electrode gap visualized by SFM, the molecule labeled 'MT' was chosen
for connection with the right electrode structure ('E'). b) The SEM was
used for the creation and imaging of an EBD line (arrows). The MTs are not
visible in the SEM contrast, the structuring was solely based on the SFM
image shown in a). c) Scanning force micrograph of the connected MT, the
written EBD line is clearly visible (cf. zoom in inset).
Discussion
MT adsorption
The presented paper focuses on steps toward the preparation of electrically
contacted biomolecules (namely, microtubules) by means of a microstructured
surface and electron beam structuring. Therefore, an adsorption of the
microtubules to the surface is essential (Fig. 1a). The adsorption of fixed
and native MTs onto various substrates (e.g., modified glass, HOPG,
modified silicon wafers) has been studied (Fritz et al., 1995; Hameroff et
al., 1989; Turner et al., 1995; Vater et al., 1995; Vinckier et al., 1995).
However, there was no detailed investigation of the materials used for the
electrode set-up in the presented study: gold and silicon oxide. MTs
without MAP2 exhibited no adsorption onto either of these materials. A
significant binding of MTs to both materials could be observed after
addition of MAP2 to the assembly solution. These microtubule-associated
proteins are known to be adhered to the outer surface of MTs. Apparently,
they enable the MT-substrate binding by a bridging effect. For contacting
applications, a controlled ad/desorption would be of special interest.
Therefore, adsorption studies were conducted by scanning force microscopy
and video-enhanced microscopy using the MAP2-dependent adsorption of MTs.
The experiments revealed that MAP2 alone adsorbs to the substrate, and that
such a modification is the prerequisite for a high affinity to MTs (in
contrast to no adsorption in the case of unmodified glass). The globular
structures revealed in scanning force micrographs of MAP2-modified surfaces
point to aggregates of MAP2. This aggregation effect does not influence the
potential to bind MTs, as demonstrated by the adsorption (Fig. 2b) after
inflow of MT solution. An interesting feature for controlled deposition of
MTs is the reversibility of their MAP2-mediated adsorption. The MAP2-MT
interaction is salt-dependent, a high ionic strength (as the 0.5 M NaCl
used in the experiment) is sufficient to induce a dissociation of the
protein-protein complex (Vallee, 1983; Vater et al., 1986). So high-salt
buffer removes MAP2-mediated bound MTs, what could be used to remove all
molecules which are not contacted after structuring of the electrodes. In
this case, the structured electrodes would arrest the contacted MT on the
substrate. An interesting feature in such a set-up is the interface area
between MT and substrate. After high salt conditions, the bridging MAP2
will be removed, and MTs contacted (and therefore fixed) on both ends could
be considered as self-supported with minimized substrate interactions.
MT orientation
For optimization of electrical contacting a parallel orientation of the
adsorbed molecules referred to the prestructured gold electrodes would be
helpful. Thereby, the probability of adsorbed molecules which connect both
electrodes by spanning over the gap could be increased. Due to the decrease
of molecules with other orientations (which disturb electrical measurements
in the case of multiple intersections) an increase in MT surface density
would be possible (e.g., by increases of adsorption time or MT
concentration), resulting in more molecules adsorbed in a desired position.
A simple method inducing a preferred MT orientation is the flow adsorption
(Turner et al., 1995). The application of this method to microstructured
substrates yielded a high percentage of MTs aligned along the flow
direction, and is especially efficient in the case of longer (5 µm and
more) MTs (Fig. 3).
MT contacting
Although the results from aligned MTs are promising, there is still the
need for a technique which allows the contacting of single molecules out of
an ensemble deposited at the substrate surface, e.g., for adjustment of
various electrode gaps, or to chose special molecules for contacting
purposes. The potential of electron beam-induced deposition (EBD) for
electrode structuring was investigated. This deposition bases on the effect
that the focused electron beam (e.g., in the SEM) induces a build-up of
material on the point where it reaches the surface. The material for the
created structure comes from the gas inside the vacuum chamber, which
results usually in carbonaceous compounds. Electrical characterization
yielded a low conductivity (Fritzsche & Porwol, unpublished results). For
the structuring of conducting electrodes the lines could be used as etching
mask of an underlying conducting layer (e.g., gold). Gold lines with
nanometer width and a high conductivity were yielded after an etching step
(Fritzsche & Porwol, unpublished results). On the other side, it is
possible to control the composition of the deposited material by
introduction of gases or gas mixtures of defined composition, what can be
used for the creation of metal lines (Koops et al., 1996).
The application of this technique in a SEM allows the structuring but also
the immediate control of the created structures (thereby providing a fast
feedback). A crucial point is that an orientation using surface features
(e.g., gold electrodes) is possible, which allows the contacting of
molecules not visible in the SEM by prescreening in a SFM and using the
scanning force micrographs. This procedure is explained by Fig. 5: An
electrode structure with adsorbed MTs is visualized by SFM (Fig. 5a). A
long MT is chosen for connection to the right electrode ('E') . This SFM
image is the base for the structuring of the EBD line. By using the known
angle and distance between the free MT end and the other electrode an EBD
line (arrows in Fig. 4b) was written using the SEM in a blind manner. Then,
the SFM was used to monitor the results of the EBD (Fig. 4c): A line
structure connecting free MT-end with the free electrode is visible,
confirming the successful contact structuring.
Conclusions
The presented results demonstrated the great potential of the proposed
approach for defined molecule adsorption and subsequent single molecule
contacting using the EBD technique. Further work will be aimed at the
improvement of the EBD-structures (especially the conductivity), and the
subsequent electrical characterization of the contacted molecules.
Acknowledgments
The authors wish to thank H. Porwol for substrate preparation; S. Jakobs,
A. Dupare, W. Vater, and M. Kittler for SFM access; S. Häfner for
excellent technical assistance with microtubule preparation; F. Jahn for
SEM measurements and EBD work; and M. Schubert for valuable discussions
about the EBD mechanism. The work was supported by the BMBF (Project-No.
120568/IMB).
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