Ultramicroscopy 91 (2002) 281–285

Patterning DNA on mm scale on mica
M. Fujitaa,b, W. Mizutanic,*, M. Gadd, H. Shigekawab,e, H. Tokumotoc
aNational Institute of Advanced Industrial Science and Technology (AIST), Nanotechnology Research Institute,
AIST Tsukuba Central 4, Higashi 1-1-1, Tsukuba 305-8562, Japanb
Institute of Applied Physics, University of Tsukuba, Tsukuba 305-8573, Japanc
Joint Research Center for Atom Technology (JRCAT)–AIST, Higashi 1-1-1, Tsukuba 305-8562, Japand
JRCAT—Angstrom Technology Partnership, Higashi 1-1-1, Tsukuba 305-0046, Japane
Core Research for Evolutional Science and Technology (CREST), Honcho 4-1-8, Kawaguchi, Saitama 332-0012, Japan
Received 15 June 2001; received in revised form 29 November 2001

Abstract
Double-stranded DNA molecules were patterned by selective adsorption to aminosilane patterns on mica surfaces.
Line patterns with 10 mm spacing were made by photolithography and transferred to a polymer stamp. The stamp wasthen used for applying aminosilane molecules by microcontact-printing technique on mica substrates. We applied DNAin Tris-EDTA (TE) buffer solution on the patterned substrate, and incubated it for 5 min at room temperature. Thesample was then rinsed with pure water, and dried with nitrogen gas. Tapping mode force microscopy showed thatDNA was adsorbed selectively on the aminosilanized parts of the mica substrate. We also tried to bridge two aluminumelectrodes with DNA using AC electrophoresis. r 2002 Elsevier Science B.V. All rights reserved.
Keywords: DNA; Aminosilane; Microcontact printing; Atomic force microscopy

1. Introduction
Microarrays of complementary DNA have beensuccessfully applied for monitoring gene expressions. High-density DNA arrays were fabricatedby ‘light-directed’ oligonucleotide synthesis [1],and patterns as small as 20 mm were utilized [2].
Another type of microarrays is fabricated without
photomasking process, but by direct application ofsmall droplets ðB0:005 mlÞ of polymerase chain
reaction (PCR) products, which spread over
100 mm and resulted in a larger pattern [3,4].
*Corresponding author.
E-mail address: w.mizutani@aist.go.jp (W. Mizutani).

Conﬁning DNA within 10 mm on a substrate could
help fabricating higher density microarrays withthe latter method.
In addition to applications like DNA chips andcomputing, DNA is expected to contribute in
future molecular electronics as a molecular wire.
DNA network structures [5–7] or nanoparticleDNA complex may present new electronic properties, e.g. combined effects of quantum interference
and single electron tunneling. For assembling
DNA with other electronic components, precise
techniques for positioning DNA and/or DNA
complexes will be needed. Since DNA was foundto adsorb selectively on aminosilane ﬁlms [6,8,9],
we utilized such property to pattern it. This was

0304-3991/02/$ - see front matter r 2002 Elsevier Science B.V. All rights reserved.
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done by creating aminosilane patterns on mica
surfaces by the microcontact printing ðmCPÞtechnique [10,11].
Most of the mCP reports used thiols on goldsubstrates, while there have been relatively few
reports [12–14] applying the technique to silaneoxide, i.e., molecules on insulator system, which
will be more practical to combine with semiconductor devices. One form of future moleculardevices will be fabricated using electrodes withnanometer-scale insulative spacings where functional molecules are to be immobilized to act as
nanodevices. Such molecules are supposed
to be selectively adsorbed in the gap, aligned
between the electrodes by, e.g., electric ﬁelds, andconnected to the surfaces of the electrodes. We will
demonstrate an example of this type of approachin the following section. It was in 2000 that Finnie
et al. studied the formation mechanism and qualityof silane self-assembled monolayers (SAMs) bymCP on Si oxide [15]. Furthermore, Finnie et al.
and Wang et al. showed that the octadecyltrichlorosilane (OTS) and docosyltrichlorosilane(DTS) patterns transferred to silicon surfaces
worked as resists against KOH etching [15,16].
Huang et al. utilized ‘selective dewetting’ ofdiazonaphthoquinone novolak polymer (photoresist) for patterned growth of carbon nanotubes[14].
Most of silane mCP studies used alkyltrichlorosilanes such as OTS and DTS, in contrast
to a few reports in which aminosilanes wereused for silanization [17]. To step forward towards
the goal of molecular scale construction,
we believe that direct chemical reaction
with the patterned SAM ﬁlms will be necessary.
In general, for constructing complex molecular
structures on target surfaces, it will be easier tobuild them from smaller soluble molecules using
surface chemical reactions than to synthesize
complex large molecules separately and adsorb
them on the surface: This is because such large
molecules are often insoluble and hard to behandled. In this work, however, we patterned
double-stranded DNA molecules using aminosilane ﬁlms on mica, since techniques for handlingDNA are well-known. With the mCP technique, wecould readily conﬁrm the selectivity of DNA

adsorption on bare and aminosilanized areas on
mica surfaces.
We further tried to control molecular orientation using an electric ﬁeld between comb shaped
aluminum electrodes. Stretched DNA molecules
were observed in the silanized areas between theelectrodes.

2. Materials and methods
Photoresist patterns of 5 and 10 mm line-andspace were prepared by photolithography on asilicon substrate which was used as a master forthe stamp. Patterned poly(dimethylsiloxane)(PDMS) stamps were made by mixing silicone
elastomer and curing agent (Dow Corning Co.,SYLGARD184), and pouring the mixture in aplastic disposable container with the silicon masterat the bottom. The container was then evacuatedto remove the bubbles, and heated in an oven at
601C for 12 h in air. Then we took the hardened
polymer out from the container, removed themaster, and cut the elastomer in a suitable shapearound the stamp. We applied 1 mM hexane
solution of 3-(2-aminoethylamino)propyltriethoxysilane (aminosilane) directly to the PDMS stamp
in a globe box ﬁlled with nitrogen gas.
Then aminosilane molecules were microcontactprinted on mica substrates for 20–30 min(Fig. 1(a)). We applied 175 mg=ml lDNA ð48:5 kbpÞon the patterned substrate (Fig. 1(b)), washedwith pure water, and dried under nitrogen gas(Fig. 1(c)).

(a)

(b)

(c)

Fig. 1. mCP aminosilane and DNA patterning scheme.

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3. Results and discussion
Tapping mode atomic force microscopy (AFM)
showed a high selectivity of DNA adsorption on
the aminosilanized patterns compared to baremica as shown in Fig. 2.
The observed high selectivity may be explainedby electrostatic interaction between DNA andsubstrate. DNA molecules are negatively charged
due to the negative charges of the phosphatebackbone in the buffer solution. The surface ofcleaved mica is also negatively charged, while theaminosilanized surface is supposed to exhibit
positive charge [5] (Fig. 1(a)), and attracts DNAs.
DNA molecules are mobile on untreated mica, andcan be easily washed off. Fig. 2 shows DNA
molecules that were strongly adsorbed to aminosilane and withstood washing and drying
processes.
As Pompe et al. called the ‘elevated rim’
structure and explained by the capillary condensation of excess solution in their article [13], theboundaries of the microcontact-printed pattern ofaminosilane tend to protrude similarly (Figs. 2(a)283and (e)). Aminosilane molecules sometimes seemto polymerize and form protruded structures asshown in Fig. 2(c). The protrusions can be bundledor globular form of DNAs [18]. In this case,uniform ﬁlms may not be formed, but aroundthose structures, aminosilane molecules are present, and even the aggregated structures seem toadsorb DNA molecules.
In Figs. 2(a) and (b), DNA molecules are seenoutside the elevated rims about 500 nm (e.g., thewhite arrow in Fig. 2(b)), probably due to thespillover of aminosilanes from the edge of thestamp. Another patterning problem can be observed in the form of tilted lines over the pattern(Fig. 2(a)). This may be formed as a result of thedisplacement of the stamp during contacting mica
surface. Careful maneuver or controllable device isrequired to improve patterning precision down to100 nm:
In addition to the precise positioning, techniques for stretching DNA have been extensively
studied [19,20]. Recently, we succeeded in orienting DNA molecules by tethering them from one
end on mica surfaces through a crosslinker andFig. 2. DNA selectively adsorbed on mCP aminosilane. (a) and (b) Height of the DNA part measures about 3 nm; indicating bundle
formation. The white arrow in (b) indicates a position where the wall along the boundary is broken and some DNAs are adsorbed
outside the patterned area. (c) Height of the DNA measures about 0:5 nm; which is typical for AFM observation of single DNAs
[6,24]. About the protrusions, see the text. (d), (e) Crosssectional proﬁles along the lines in AFM images (a) and (c), respectively.

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Fig. 3. DNAs bridging Al electrodes 5 mm apart. In addition to the stretched DNA strands, there remain some unspeciﬁc structures,
which may be by-products due to some electrochemical reaction.

stretching them by shear force [20]. In this work,
we tried to bridge two electrodes with DNA using
AC electrophoresis (Fig. 3(a)). We used interdigitated electrodes with a spacing of 5 mm which werefabricated on mica using the lift-off process. Afteraminosilanizing the mica substrate, we covered thecomb electrode with DNA solution, and appliedan AC voltage of 5 V at 1 MHz which produces
the same electric ﬁeld as reported previously by
Washizu et al. [21,22] for 30 s:
After drying the buffer in air at room temperature, large structures (most probably, salt crystals)
appeared, which seemed to cover DNA. On
washing the sample with Milli-Q water, DNA
could be observed bridging the two electrodes(Fig. 3(b)). Even after washing, the surface waspartially covered with unspeciﬁc structures, which
could not be removed by the rinsing. We speculatethat they could be by-products due to someelectrochemical reaction.
DNA stretched by electric ﬁeld was also
observed using AFM by Ueda et al. [23]. In theirexperiment, they immobilize DNA molecules on
the slope of Al electrodes. Our result shows thatthe DNA can be also immobilized ﬁrmly on an
insulative gap area, e.g., a silanized mica surface,with the orientation parallel to the ﬁeld.

4. Conclusion
We applied microcontact printing technique topattern organic thin ﬁlms on mica surfaces.

10 mm patterns made by photolithography weretransferred to a silicone rubber stamp, and thenaminosilane molecules were microcontact-printed
on mica substrates. We applied DNA in TE buffer
solution on the patterned substrates, and foundthat the DNA was adsorbed selectively on theaminosilanized areas with a precision of about
500 nm: We could orient DNA molecules byapplying an electric ﬁeld. Stretched molecules wereimmobilized on the aminosilanized mica substrate.
Some DNA molecules remained ﬁrmly attached tothe silanized areas after rinsing the sample with
Milli-Q water.

Acknowledgements
We are grateful to Dr. K. Umemura for goodsuggestions and variable comments. This work
was supported by the New Energy and Industrial
Technology Development Organization (NEDO)
of Japan.

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