ISSN 1068-3356, Bulletin of the Lebedev Physics Institute, 2007, Vol. 34, No. 12, pp. 366–371.

c Allerton Press, Inc., 2007.
Original Russian Text
c G.K. Chudinova, I.A. Nagovitsyn, M.A. Kononov, I.A. Maslyanitsyn, V.D. Shigorin, V.V. Savranskii, 2007, published in Kratkie Soobshcheniya
po Fizike, 2007, Vol. 34, No. 12, pp. 43–51.

Atomic-Force Microscopy Studies of the Complexes of DNA

and Single-Wall Carbon Nanotubes
G. K. Chudinova, I. A. Nagovitsyn, M. A. Kononov,
I. A. Maslyanitsyn, V. D. Shigorin, and V. V. Savranskii
Prokhorov General Physics Institute, Russian Academy of Sciences,
ul. Vavilova 38, Moscow, 119991 Russia
Received November 19, 2007

Abstract—The interaction of DNA samples extracted from tobacco leaves of two types with carbon
nanotubes (CNTs) was studied. Atomic-force microscopy images of DNA–CNT complex films were
obtained. The possibility of forming ordered self-assembling structures in a DNA–CNT film was
shown.
DOI: 10.3103/S1068335607120068

Recently, carbon nanotubes attract close attention, in particular, as biocompatible materials with
biological and medical applications. Carbon nanotube-based bionanocomposites can be applied to
(i) biosensor production; (ii) synthesis of molecular structures for vaccine and drug transport to a certain
human organ; (iii) the use of nanotubes as a cell growth matrix; (iv) the development of bioelectronic
devices which can be implanted into the human organism [1–5].
The use of nanotubes in the development of a sensitive element of biosensors leads to appreciable
advantages, specifically, a probably sharp sensitivity enhancement, a decrease in the sensitive element
size due to a developed surface, and an increase in the measurement rate and reproducibility [6–13].
Recently, atomic-force microscopy (AFM) is successfully used as a detecting element of a sensor.
This method has giant potential for both quantitative and qualitative determination of biomaterials. AFM
is a very accurate instrument for characterizing the image of complex biomolecular systems. Carbon
nanotubes (CNTs) represent an ideal material for these purposes. Recently, AFM in the dynamic mode
is used for chemical and molecular-biological analyses of DNA and not only DNA [13–27].
This study is the first among those of the planned series with the overall aim to perform a systematic
study on the choice of optimum conditions of nanotube-based bionanocomposite fabrication for the
sensitive element of the biosensor.
Experimental. Single-wall raw nanotubes (NTs) synthesized by the technique [28] were used; the
nanotube content in the raw product was 20%.
DNA samples (DNA1 and DNA2) were extracted from leaves of two tobacco sorts of genuine
Nicotiana Tabacum L. by the technique [29]. The initial DNA concentrations were 1.5 · 10−6 M (DNA1)
and 3.3 · 10−6 M (DNA2).
It is known from the literature that the extraction of nanotubes themselves from a reaction mixture is
performed using solutions of surfactants such as triton Х-100 with various concentrations [30, 31]. In
this study, DNA molecules themselves were used as an active material for NT extraction from the initial
product.
DNA–NT complexes were obtained in water (pH 6.0) and in a 0.05-M carbonate- bicarbonate buffer
(рН 9.2) containing sodium chloride with a concentration of 0.02 M. The experiment was carried out as
follows: four weighed portions of raw NTs (2 mg) were poured with hot solutions (95◦ C) containing
DNA1 or DNA2 in amounts of 200 µl in 6 ml of water or buffer. The prepared samples were exposed to
ultrasound using an UZDN-F device for 90 min. The DNA–NT solutions were cooled during ultrasound
processing using a mixture of ice, sodium chloride, and water (the initial temperature was –7◦ C). The
DNA and NT temperature during this process was on average 35◦ C. The prepared suspension was
centrifuged for 20 min at 2000 rpm using an OP-8UKhL4.4 centrifuge. The solution containing the
DNA–NT complex was placed using a micropipet (with a maximum volume of 200 µl) onto polished

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 ATOMIC-FORCE MICROSCOPY 367

Fig. 1. (a) Absorption spectra of initial concentrated solutions of DNA1 and DNA2 before mixing with nanotubes;
(b) absorption spectra of DNA1 and DNA2 with nanotubes after ultrasonic processing (200 µl of initial concentrated
solution of DNA1 or DNA2 + 2 mg of raw nanotubes in 6 ml of 0.05-M buffer solution).

quartz substrates (35×10×1.5 mm in size) preliminarily placed into Petri dishes. Plates were dried for a
day in closed Petri dishes at room temperature.
The absorption spectra of initial DNA solutions, DNA–NT complex solutions, and films on quartz
plates were measured using a Shimadzu UV 250 spectrophotometer. The film structure was studied
using a Р4-SPM-MDT microscope (ZAO “NT-MDT” Company, Russia) in the force scanning mode
using the P7− SPM program.
Results and discussion. Figure 1 shows the absorption spectra of initial concentrated DNA1 and
DNA2 solutions (a) and the absorption spectra of the obtained DNA complex with NTs after ultrasonic
processing in solution (b). Figure 2 shows the absorption spectra of initial DNA samples and DNA
complexes with NTs on quartz substrates after ultrasonic processing and drying in air. As can be
seen, the absorption spectrum maximum of the DNA complex with NTs redshifts by 15–20 nm with
respect to the DNA absorption maximum both in solution (Fig. 1) and on the quartz substrate (Fig.
2). This suggests that the shift of the DNA absorption band maximum during the interaction with NTs
is explained exactly by the formation of the DNA complex with NTs, rather than by a change in the
environment during the film formation.
The electron microscopy image of raw NTs (the initial product containing NTs) is shown in Fig. 3
(before ultrasonic extraction). We can see the amorphous structure of the initial sample. Because of
the small sizes of NTs themselves [32–34], AFM imaging of the surface consisting of NTs has some
difficulties. Figure 4 shows the AFM image of film grown by evaporation of DNA1 solution without
NT addition on a quartz plate (control experiment). It is easily seen that the surface consists of densely
packed ball structures which diameter varies from 30 to 60 nm.
Figures 5 and 6 show the images of NT films with DNA1 and DNA2, respectively, obtained by
evaporating an aqueous solution at рН 6.0 after ultrasonic processing. We can see an essential difference
in both size and shape of the structures formed in the films (Figs. 5 and 6) in comparison with the single-
component DNA1 film structure (Fig. 4). The film structures of the DNA1 complexes with NT (Fig. 5)
remind a spruce branch; the size of a single domain (“branch”) is 300 nm and larger. The structure of
films containing DNA2 complexes with NTs represent aggregates of ball (100–120 nm) and oval (150–
350 nm) structures (Fig. 6).
Quite different images are characteristic of DNA1 and DNA2 complexes with NTs, obtained by
evaporating a buffer solution (рН 9.2, in the presence of 0.02-M sodium chloride), which are shown
in Figs. 7 and 8. For DNA1 (Fig. 7), highly ordered cord-like structures uniformly distributed over
the surface are observed. The cord width is about 100 nm, which does not allow us to suggest that
there is only one single-wall NT in the DNA1 complex with CNT, braided with the DNA1 molecule
(the schematic diagram of the complex is shown in [35, 36]). The determination of the structure of the

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Fig. 2. Absorption spectra of two DNA samples without nanotubes (1 and 3) and DNA complexes with nanotubes (2
and 4) on the quartz substrate.

Fig. 3. Electron microscopy image of raw nanotubes. The product contains 20% of nanotubes.

Fig. 4. AFM image of the DNA1 film (without nanotubes).

Fig. 5. AFM image of the DNA1 film with nanotubes, obtained by evaporating an aqueous solution.

obtained complex requires further study. We note that atomic-force scanning of the quartz plate shows
inhomogeneity of the obtained film in all cases. This can be caused, first, by that it is impossible to
prepare a uniform film by solution evaporation; second, as this is often observed for Langmuir films,
the film structure represents domains with voids between them, the so-called defects. A decrease in

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 ATOMIC-FORCE MICROSCOPY 369

Fig. 6. AFM image of the DNA2 film with nanotubes, obtained by evaporating an aqueous solution.

Fig. 7. AFM image of the DNA1 film with nanotubes, obtained by evaporating a buffer solution (рН = 9.2).

defects of such a film by the approach of domains results in film destabilization (destruction); probably,
such a structure of domain and voids alternation is thermodynamically favorable [37–40]. However,
self-assembling of DNA1 complexes with CNTs during solution evaporation is the fact. Similar highly
ordered systems can be further used in the development of sensitive elements of sensor systems.
In the case of DNA2 (рН=9.2), filamentary structures are formed (Fig. 8a). Magnification of the
image (Fig. 8b) shows that filamentary structures are composed of ball domains 100 nm in size and
larger. We assume that aggregation of DNA2 complexes and nanotubes occurs in observed domains
(aggregation of nanotubes was demonstrated in [41] by AFM). We note that ball [42] and filament
structures are described in the literature for various DNA types [43].
Thus, by the example of DNA samples extracted from leaves of tobacco of two different sorts, it
was shown that the difference in the nucleotide composition of DNA has an effect on the shape of
aggregates of DNA complexes with CNTs formed on the quartz plate surface. It was shown that self-
assembled ordered domains consisting of DNA–CNT complexes are formed on the quartz plate surface.

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370 CHUDINOVA et al.

Fig. 8. AFM image of the DNA2 film with nanotubes, obtained by evaporating a buffer solution (рН = 9.2).

Preliminary results were also obtained, which show that conditions of preparation (рН, buffer, presence
of salt) of the complex also affect its shape.

ACKNOWLEDGMENTS
The authors are grateful to E.D. Obraztsova (Natural Science Center, Prokhorov General Physics
Institute, Moscow) for nanotubes put at their disposal, I.P. Beletskii (Institute of Theoretical and
Experimental Biophysics, Pushchino, Moscow Oblast) for DNA put at their disposal, and N.D. Vasilieva
(Moscow Power Energy Institute) for electron-microscopy imaging.
This study was supported by the Russian Foundation for Basic Research, project no. 07-02-00160-a.

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