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Making Electrical Contact to Single Molecules

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: , FAX 0049-3641-657744

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

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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.


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


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 (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.


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 The fast scan direction (left to right) is thereby assigned as 0°, from which the measured angles opened counterclockwise.


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).


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.


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.


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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