In the study a new biosensor contained an oligonucleotide-carbon nanotubes-stearic acid monocrystalline
clusters in its sensitive coating has been developed. The electrochemical characteristics of sensors with
different coatings distinguishing by content of oligonucleotide and cerium ions have been measured. It was
found that the presence of Ce in the coating leads to decrease of sensor capacity changes at measurements
in mediums of different content. The oligonucleotides anchored in sensor coating attenuate cell nonantisense
adhesion to sensor surface.
When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/3547

Egorov, A.S.

Egorova, V.P.

Govorov, M.I.

Grushevskaya, H.V.

Krylova, N.G.

Kulahava, T.A.

Lipnevich, I.V.

Orekhovskaya, T.I.

Semenkova, G.N.

Shulitsky, B.G.

Ulashchik, V.S.

Vasiljev, N.V.

SocioEconomic Challenges Journal (Sumy State University)

PROCEEDINGS OF THE INTERNATIONAL CONFERENCE  
NANOMATERIALS: APPLICATIONS AND PROPERTIES 
Vol. 2 No 4, 04NABM22(4pp) (2013) 
 
 
2304-1862/2013/2(4)04NABM22(4) 04NABM22-1  2013 Sumy State University 
Cellular Adhesion to Carbon Nanotubes-anchored Oligonucleotide:  
Electro-chemical Detection 
 
H.V. Grushevskaya1,*, N.G. Krylova1,†, I.V. Lipnevich1, T.I. Orekhovskaya2,A.S. Egorov3, V.P. Egorova4, 
M.I. Govorov3, T.A. Kulahava1, G.N. Semenkova1, B.G. Shulitsky2, V.S. Ulashchik3, N.V. Vasiljev1 
1 Belarusian State University, 4 Nezavisimosti Ave., Minsk, 220030, Belarus 
2 Belarusian State University of Informatics and Radioelectronics, 6 P. Brovki Str., Minsk, 220013, Belarus 
3 Institute of Physiology, Nat. Acad. Sci. of Belarus, Academicheskaya St. 28, 220072, Minsk 
4 Belarusian State Pedagogical University, Sovetskaya St. 18, 200005, Minsk 
 
(Received 12 June 2013; published online 01 September 2013) 
 
In the study a new biosensor contained an oligonucleotide-carbon nanotubes-stearic acid monocrystal-
line clusters in its sensitive coating has been developed. The electrochemical characteristics of sensors with 
different coatings distinguishing by content of oligonucleotide and cerium ions have been measured. It was 
found that the presence of Ce in the coating leads to decrease of sensor capacity changes at measurements 
in mediums of different content. The oligonucleotides anchored in sensor coating attenuate cell non-
antisense adhesion to sensor surface. 
 
Keywords: Oligonucleotides, Multi-walled carbon nanotubes, Electrochemical biosensor, Impedance spec-
troscopy 
 
  PACS numbers: 87.85.fk, 87.85.Qr 
 
 
                                                        
* grushevskaja@bsu.by 
† nina-kr@tut.by 
1. INTRODUCTION 
 
Impedance analysis of biological objects is the prom-
ising method for the development of biomedical diag-
nostic test systems [1]. Impedance monitoring of cell 
monolayer grown or adhered to nanobiosensors is a 
cheap, practical and non-invasive method of quantita-
tive estimation of cell proliferation, cell-cell communi-
cation and cellular functional activity at the influence 
of different chemical compounds, as well as for the 
evaluation of their cytotoxic effects [2-5].  
Nucleic acids offer some advantages, which include 
combinatorial encoding of interactions, easiness of syn-
thesis and modification, as well as minimal interaction 
with cell membrane molecules. DNA programmed ad-
hesion is also strong, rapidly formed and is reversible 
upon addition of DNase [6-7]. DNA-based attachment 
strategy is widely developed for the generation of arti-
ficial tissues and the incorporation of living cells into 
device settings.  
The cells with membrane ss-DNA- or ss-oligonucleo-
tides linking to the complimentary ss-DNA- or ss-
oligonucleotide modified surface is used to direct the 
physical interactions between cells and substrates in-
dependent of the adhesion machinery. Cells fixed on a 
surface interact weakly with the substrate and cell-cell 
communication is insignificant. Currently, there are no 
developed electrochemical techniques to detect func-
tional changes that occur in cells fixed near the surface 
of a sensor at their stimulation or destruction. Regis-
tration of such weak signals demands a high sensitivity 
of a sensor. With this in mind, the improvement of the 
method for express electrochemical measurement of 
cellular parameters is of great interest.  
The purpose of the study is to develop the electro-
chemical impedance sensor that contains in a sensing 
coating the oligonucleotide sequence, anchored on mod-
ified carbon nanotubes (CNT) and to evaluate non-
antisense effect of anchored CNT-oligonucleotides com-
plexes on C6 rat glioma cells. 
 
2. MATERIALS AND METHODS 
 
In this work a system of electrode pairs with inter-
digital structure has been used. Each pair located on a 
glassceramics substrate represents itself an "open type" 
electrical capacity.  
Dielectric coating of sensor aluminum electrodes 
represents a layer of porous anodic aluminum oxide 
with pores of 10 nm diameter on which five metal-
contained Langmuir-Blodgett (LB) monolayers of thio-
phene derivatives oligomer 3-hexadecyl-2,5-
di(thiophen-2-yl)-1H-pyrrole (HDTP) [8] and two LB-
monolayers of multi-walled carbon nanotubes (MCNTs) 
were sequentially deposited. MCNT with diameters 
ranging from 2.5 to 20 nm and a length of 2.5 μm were 
covalently functionalized with carboxyl groups and 
non-covalently modified with stearic acid molecules [9]. 
The oligonucleotide ON1 (structure of the sequence: 5’-
GCCATATACTCTCCTTGGTGACA-3’) was synthesized 
at OOO Primetex (Minsk). 
To obtain LB-monolayers with oligonucleotide-
MCNT complexes, alcoholic solution of oligonucleotide- 
MCNT complexes were mixed with a solution of stearic 
acid in hexane. The resulting mixture was homoge-
nized to form reverse micelles of stearic acid with oli-
gonucleotide-MCNT complexes inside it [10]. 
HDTP LB-films formed on the subphase containing 
iron (Fe(NO3)3) and/or Ce (Ce2(SO4)3), concentration 
ratio of [Ce] to [Fe] in subphase was equial to 0 or 0.01. 
LB-sublayers of MCNT or MCNT-oligonucleotide 
complexes have been formed on subphases with or 
without Ce. 
 
Measurements were performed in a cuvette with a 
buffer medium. Earle's balanced salt solution consisted 
 H.V. GRUSHEVSKAYA, ET AL. PROC. NAP 2, 04NABM22 (2013) 
 
 
04NABM22-2 
of 0,12 M NaCl, 5,4 mM KCl, 0,9 mM NaHPO4·2H2O, 
0,8 mM MgSO4·7H2O, 2,5 mM CaCl2, 5,6 mM glucose, 
26,2 mM NaHCO3 (“Analys Х”, Belarus) was used. 
All other reagents were of analytical grade. All 
aqueous solutions were made with bidistilled deionized 
water. 
C6 rat glioma cells obtained from culture collection 
of Institute of Epidemiology and Microbiology (Minsk) 
were grown on a sensor surface in a Dulbecco's modi-
fied Eagle medium (DMEM) supplemented with 10 % 
fetal bovine serum and 1·10-4 g/ml gentamycin at 37 °C 
in a humidified 5 % CO2 atmosphere. 
Registration of the sensor characteristics was car-
ried out by impedance spectroscopy. Interdigital sen-
sors were connected into the pulse-adjustable RC-
autooscillator. Electrochemical measurements were 
carried out in auto-oscillate regime at a frequency of 
quasi-resonance [11]. 
Microdiffraction patterns and electron microscopic 
images were obtained by means of transmission elec-
tron microscope JEM-100CX (JEOL, Japan) (TEM) at 
an accelerating voltage of 100 kV. Structural analysis 
have been performed on the objects previously deposit-
ed on a copper grid with a formvar polymer coating. 
Visualization of the cellular monolayer was carried 
out using a confocal micro-Raman spectrometer Nano-
finder HE («LOTIS-TII», Tokyo, Japan). 
 
3. RESULTS AND DISCUSSION  
 
3.1 Oligonucleotide-MCNT LB-complex  
structure 
 
Carbon nanotubes form highly ordered LB-CNT-
clusters in the LB-monolayers. According to the TEM 
images shown in Fig. 1a, a diameter of MCNTs in LB-
cluster equals to 3 nm. Fig. 1b demonstrates that the 
oligonucleotide-MCNTs LB-complexes represent them-
self MCNTs covered close-packed layer of oligonucleo-
tide molecules, nucleotide layer thickness is of the order 
of 2 to 10 nm. 
Highly oriented MCNTs arrays are formed in the ep-
itaxial growth on an iron-containing crystalline LB-
films of HDTP [10]. It has been  shown that the diffrac-
tion patterns of LB-nanotube clusters on the HDTP LB-
films are characterized by Kikuchi lines [10] being 
characteristic for mono-crystallites with size of 0.5 –
 1 μm. 
The oligonucleotide molecules located on the MCNT 
in LB-film are in a highly ordered state. In the zero-
node of the reciprocal lattice a diffraction pattern (Laue 
pattern) of the LB-monolayers consisting of oligonucleo-
tide-MCNT-stearic acid mixture represents itself a 
three reflexes with the class of symmetry C2. This indi-
cates that the non-covalently functionalized by stearic 
acid MCNT-oligonucleotide complexes placed on HDTP 
LB-films are also in the monocrystal form. The oligonu-
cleotide crystal interplanar spacing dp in the direction 
being orthogonal to the surface of MCNT is equal to 
0.438 nm. 
 
3.2 Electrochemical impedance spectroscopy 
 
The sensors have been tested for 6 different types of 
coatings: 1) iron-containing HDTP LB-film and two LB-
MCNT-monolayer without Ce (coating 1a) with oligonu-
cleotide (coating 1b), 2) Fe-and Ce-containing HDTP LB-
film and two LB-MCNT-monolayer without Ce (coating 
2a) with oligonucleotide (the coating 2b), 3) Fe-
containing HDTP LB-film and the two Ce-containing 
LB-MCNT-monolayer (coating 3a) with oligonucleotide 
(coating 3b). 
 
 
 a 
 
 
 b 
 
Fig. 1 – TEM image of two-monolayer MCNTs-stearic acid mix-
ture LB-films formed on Ce2(SO4)3 salt aqueous solution (а) and 
deposited on three HDTP LB-monolayers formed on Fe(NO3)3 
salt aqueous solution with pH finished by hydrochloric acid;  
(b) TEM image of two-monolayer MCNTs-oligonucleotide com-
plexes-stearic acid mixture LB-film deposited on five-
monolayer iron-containing HDTP LB-film 
 
In bidistilled water, the dielectric properties of LB-
MCNT-films and oligonucleotide-MCNT complexes in 
LB-films appear as a change of capacity of the double-
charged Helmholtz layer which is formed at the inter-
phase boundary between insulating barrier layer of an-
odic aluminium oxide and water. At low frequency range 
200 – 400 kHz a sharp decrease of capacity of the sensor 
placed into electrolyte has been registered, which indi-
cates the presence of a significant ionic component of the 
double layer capacity. At the higher frequency range 
(600 – 1300 kHz), the capacity of sensor varies slightly 
due to the large distribution of relaxation times of ions 
(H+, OH–) in the pores and small dipole relaxation times. 
The measurements of electrical capacity of the sensor 
with deposited LB-MCNT-films showed that a change of 
capacity of the sensors with and without oligonucleotide 
has opposite sign with respect to sensor without MCNTs. 
The capacity change for sensors with LB-MCNT-cluster 
coating noncontaining oligonucleotide equals to –0.8 pF 
in the frequency range 500 – 900 kHz, whereas for sen-
sors with LB-MCNT-oligonucleotide-clusters, the capaci-
ty change equals to  +(0.8 – 1.2) pF. 
 CELLULAR ADHESION TO CARBON NANOTUBES- … PROC. NAP 2, 04NABM22 (2013) 
 
 
04NABM22-3 
Let us estimate the sensor capacity change when the 
ion concentration in the medium varies. According to the 
Maxwell-Garnett law the capacity of sensor upon its 
immersion in a conducting medium increases with the 
increase of ion concentration in the medium. The mini-
mal sensor capacity is observed for 0.15 M NaCl. If the 
sensor were immersed in Earle's medium (pH = 7.4) that 
does not contain CaCl2, then its capacity increased by 
65 ± 35 pF, 180 ± 20 pF and 90 ± 10 pF, that in the per-
centage ratio was 6 ± 4 %, 22 ± 5 % and 10,7 ± 0,9 % of 
the capacity of sensor in the Earle's medium without 
CaCl2 for coatings type 2b, 1b and 3b, respectively. In 
Earle's medium with 2.5 mM CaCl2 a further increase of 
capacity by 33 ± 10 pF (3,3 ± 1,1 %) was observed, re-
gardless of a coating type. 
For coatings that does not contain oligonucleotide, 
the capacity of sensor, which is in the Earle's medium, is 
higher by 235 ± 95 pF (21 ± 4 %) than in 0.15 M NaCl. 
For coating 1a, the capacity of sensor, which is in the 
Earle's medium, is higher by 66 ± 11 pF (6,0 ± 0,5 %) 
than in Earle's medium without CaCl2. The correspond-
ing recordings for sensors with coating 2a and 3a were 
117 ± 27 pF (10,7 ± 0,5 %) and 80 ± 21 pF (8,2 ± 0,5 %). 
 
3.3 Morphology of cells in monolayer 
 
By means of light microscopy the study of morpholog-
ical characteristics of cells in a monolayer formed on the 
sensors on the fourth day of cell growth in culture has 
been performed. It is found that the number of cells on 
the coating 2b is less than a number of cells on the coat-
ings 1b and 3b. As Fig.2 shows, morphology of cells de-
pends on the coating type. On the coating 1b the popula-
tion of cells is heterogeneous: there are both star-shaped 
cells with a high degree of flatness and having a large 
number of cell-cell contacts and cells being rounded and 
slightly flattened. On the coating 3b cells are strongly 
flattened and mainly elongated. Each cell has no more 
than three cell-cell contacts at the ends of highly elon-
gated threadlike pseudopodia. Cells on the coating 2b 
are rounded, with a small area of contact to the surface, 
processes are short, cell-cell contacts are few. 
 
3.4 Dielectric spectroscopy of cellular  
monolayer 
 
Previously it has been shown that the formation of 
the cellular monolayer on the sensor surface with coat-
ings 1a – 3a containing no oligonucleotide is accompanied 
by arising of a Helmholtz double layer electric field 
shielding. The shielding becomes apparent as sensor ca-
pacity decreasing on 1 – 3 days of cell growth and de-
pends on monolayer density and a degree of intracellular 
communication [12]. It has been found that for sensors 
with coatings 1b – 3b containing oligonucleotide, the sen-
sor capacity was increasing during the time of cell 
growth. It is stipulated by a Coulomb repulsion between 
negatively charged phosphate groups of oligonucleotide 
and negatively charged hyaluronic acid residues in cellu-
lar glycocalyx, obstructing the non-antisense cell adhe-
sion to oligonucleotide-contained coatings. 
As Fig. 3 shows, for coating 1b the sensor capacity 
decrement in regard to capacity of sensor without cells 
varies insignificantly on the second and third day of cel-
lular growth as compared with the first day of growth.  
 
 a 
 
 
 b 
 
 
 c 
 
Fig. 2 – Light microscopy images of cells grown on coating 1b 
(a), coating 2b (b) and coating 3b (c) 
 
The compensation of two processes is observed, name-
ly the capacity increase due to the rise of the number of 
cells and the capacity decrease due to the shielding effect, 
that is enhanced at establishing of cell-cell contacts. For 
the coating 2b the capacity increase on the first day of 
cellular growth is less by 200 pF, as compared with coat-
ings 1b and 3b. This indicates that the number of cells 
adhered to the coating 2b is smaller than number of cells 
adhered to the coatings 1b and 3b. For the coatings 2b 
and 3b the rise of the sensor capacity on the 2nd and 3rd 
day of cell growth demonstrates a low rate of intracellu-
lar contacts formation. These data agree with the data of 
cell morphology. 
The experimental results obtained in the work are ev-
idence of the following. First, the positively charged ions 
Ce4+ in the HDTP LB-film of coating 2b hydrolyzed oligo-
nucleotides [13]. This is accompanied by breaking of  - 
stacking interaction in oligonucleotide monocrystal fol-
lowing formation of double helices carrying a large nega-
tive charge. The negatively charged double-stranded oli- 
 H.V. GRUSHEVSKAYA, ET AL. PROC. NAP 2, 04NABM22 (2013) 
 
 
04NABM22-4 
 
Fig. 3 – Dependency of sensor capacity decrement on cell 
growth time for sensor capacity with cellular monolayer. The 
coatings of sensor used were coating 1b (triangle), coating 2b 
(square), coating 3b (rhomb) 
 
gonucleotide prevents non-specific adhesion of 
negatively charged cells due to strong Coulomb 
repulsion. 
Second, oligonucleotide hydrolysis is absent for Ce3+-
containing MCNT-oligonucleotide clusters of coating 3b. 
Therefore, - stacking interaction in the oligonucleotide 
monocrystal leads to the transfer of electrical charge 
along the - coupling chain which shields the Coulomb 
field of the negatively charged phosphate groups. 
The capacitive sensor  characteristics testify a physio-
logical state of cells in different medium. The dielectric 
properties of the cellular monolayer depend on the 
transmembrane potential of cells and are determined by 
the state of ion channels. It was revealed that the capaci-
ty of sensors with cellular monolayer depends on the 
composition of the measuring medium. It is shown that 
the capacity of sensor with cellular monolayer increases 
as follows: DMEM <Earle's medium (pH = 7.4) <Earle's 
medium (pH = 7.4) without 2.5 mM CaCl2 <0.15 M NaCl. 
The capacity increases by 150 ± 62 pF (4,3 ± 1,1 %) for 
coatings 2b and 3b, and by 300 ± 50 pF (9 ± 2 %) for coat-
ing 1a when cellular monolayer is transfered from Earle's 
medium to Earle's medium without CaCl2. Capacity of 
sensors with cellular monolayer formed on the surfaces 
that do not contain oligonucleotide, increases by 14 ± 4 % 
for coatings 1a and 2a at changing the Earle's medium on 
the Earle's medium without CaCl2. 
 
4. CONCLUSIONS 
 
Thus, a nanobiosensor having an oligonucleotide-
MCNT clusters layer in its sensitive coating has been 
developed. The sensor has a high-ordered mono-
crystalline structure of LB-MCNT-clusters and a close-
packed oligonucleotide layer. Such type of oligonucleo-
tide deposition is promising for high-sensitive detection 
of antisense oligonucleotide-bonded biological objects 
by means of electrochemical analysis due to the dis-
tance to the object must be about pair nucleotides size.  
The oligonucleotide anchored in sensor coating at-
tenuates cell non-antisense adhesion to sensor surface 
resulting in capacity increasing during cell growth. The 
presence of Ce in the sensor coating leads to prevent 
non-specific cellular adhesion. 
 
AKNOWLEDGEMENTS 
 
The present work was developed under the auspices 
of Belarusian State Program of Scientific Research «In-
terdisciplinary scientific research, new incipient tech-
nologies as a basis of stable innovative progress». 
 
 
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Cellular Adhesion to Carbon Nanotubes-anchored Oligonucleotide: Electro-chemical Detection

Electronic Sumy State University Institutional Repository

PROCEEDINGS OF THE INTERNATIONAL CONFERENCE  NANOMATERIALS: APPLICATIONS AND PROPERTIES Vol. 2 No 4, 04NABM22(4pp) (2013)   2304-1862/2013/2(4)04NABM22(4) 04NABM22-1  2013 Sumy State University Cellular Adhesion to Carbon Nanotubes-anchored Oligonucleotide:  Electro-chemical Detection  H.V. Grushevskaya1,*, N.G. Krylova1,†, I.V. Lipnevich1, T.I. Orekhovskaya2,A.S. Egorov3, V.P. Egorova4, M.I. Govorov3, T.A. Kulahava1, G.N. Semenkova1, B.G. Shulitsky2, V.S. Ulashchik3, N.V. Vasiljev1 1 Belarusian State University, 4 Nezavisimosti Ave., Minsk, 220030, Belarus 2 Belarusian State University of Informatics and Radioelectronics, 6 P. Brovki Str., Minsk, 220013, Belarus 3 Institute of Physiology, Nat. Acad. Sci. of Belarus, Academicheskaya St. 28, 220072, Minsk 4 Belarusian State Pedagogical University, Sovetskaya St. 18, 200005, Minsk  (Received 12 June 2013; published online 01 September 2013)  In the study a new biosensor contained an oligonucleotide-carbon nanotubes-stearic acid monocrystal-line clusters in its sensitive coating has been developed. The electrochemical characteristics of sensors with different coatings distinguishing by content of oligonucleotide and cerium ions have been measured. It was found that the presence of Ce in the coating leads to decrease of sensor capacity changes at measurements in mediums of different content. The oligonucleotides anchored in sensor coating attenuate cell non-antisense adhesion to sensor surface.  Keywords: Oligonucleotides, Multi-walled carbon nanotubes, Electrochemical biosensor, Impedance spec-troscopy    PACS numbers: 87.85.fk, 87.85.Qr                                                           * grushevskaja@bsu.by † nina-kr@tut.by 1. INTRODUCTION  Impedance analysis of biological objects is the prom-ising method for the development of biomedical diag-nostic test systems [1]. Impedance monitoring of cell monolayer grown or adhered to nanobiosensors is a cheap, practical and non-invasive method of quantita-tive estimation of cell proliferation, cell-cell communi-cation and cellular functional activity at the influence of different chemical compounds, as well as for the evaluation of their cytotoxic effects [2-5].  Nucleic acids offer some advantages, which include combinatorial encoding of interactions, easiness of syn-thesis and modification, as well as minimal interaction with cell membrane molecules. DNA programmed ad-hesion is also strong, rapidly formed and is reversible upon addition of DNase [6-7]. DNA-based attachment strategy is widely developed for the generation of arti-ficial tissues and the incorporation of living cells into device settings.  The cells with membrane ss-DNA- or ss-oligonucleo-tides linking to the complimentary ss-DNA- or ss-oligonucleotide modified surface is used to direct the physical interactions between cells and substrates in-dependent of the adhesion machinery. Cells fixed on a surface interact weakly with the substrate and cell-cell communication is insignificant. Currently, there are no developed electrochemical techniques to detect func-tional changes that occur in cells fixed near the surface of a sensor at their stimulation or destruction. Regis-tration of such weak signals demands a high sensitivity of a sensor. With this in mind, the improvement of the method for express electrochemical measurement of cellular parameters is of great interest.  The purpose of the study is to develop the electro-chemical impedance sensor that contains in a sensing coating the oligonucleotide sequence, anchored on mod-ified carbon nanotubes (CNT) and to evaluate non-antisense effect of anchored CNT-oligonucleotides com-plexes on C6 rat glioma cells.  2. MATERIALS AND METHODS  In this work a system of electrode pairs with inter-digital structure has been used. Each pair located on a glassceramics substrate represents itself an "open type" electrical capacity.  Dielectric coating of sensor aluminum electrodes represents a layer of porous anodic aluminum oxide with pores of 10 nm diameter on which five metal-contained Langmuir-Blodgett (LB) monolayers of thio-phene derivatives oligomer 3-hexadecyl-2,5-di(thiophen-2-yl)-1H-pyrrole (HDTP) [8] and two LB-monolayers of multi-walled carbon nanotubes (MCNTs) were sequentially deposited. MCNT with diameters ranging from 2.5 to 20 nm and a length of 2.5 μm were covalently functionalized with carboxyl groups and non-covalently modified with stearic acid molecules [9]. The oligonucleotide ON1 (structure of the sequence: 5’-GCCATATACTCTCCTTGGTGACA-3’) was synthesized at OOO Primetex (Minsk). To obtain LB-monolayers with oligonucleotide-MCNT complexes, alcoholic solution of oligonucleotide- MCNT complexes were mixed with a solution of stearic acid in hexane. The resulting mixture was homoge-nized to form reverse micelles of stearic acid with oli-gonucleotide-MCNT complexes inside it [10]. HDTP LB-films formed on the subphase containing iron (Fe(NO3)3) and/or Ce (Ce2(SO4)3), concentration ratio of [Ce] to [Fe] in subphase was equial to 0 or 0.01. LB-sublayers of MCNT or MCNT-oligonucleotide complexes have been formed on subphases with or without Ce.  Measurements were performed in a cuvette with a buffer medium. Earle's balanced salt solution consisted  H.V. GRUSHEVSKAYA, ET AL. PROC. NAP 2, 04NABM22 (2013)   04NABM22-2 of 0,12 M NaCl, 5,4 mM KCl, 0,9 mM NaHPO4·2H2O, 0,8 mM MgSO4·7H2O, 2,5 mM CaCl2, 5,6 mM glucose, 26,2 mM NaHCO3 (“Analys Х”, Belarus) was used. All other reagents were of analytical grade. All aqueous solutions were made with bidistilled deionized water. C6 rat glioma cells obtained from culture collection of Institute of Epidemiology and Microbiology (Minsk) were grown on a sensor surface in a Dulbecco's modi-fied Eagle medium (DMEM) supplemented with 10 % fetal bovine serum and 1·10-4 g/ml gentamycin at 37 °C in a humidified 5 % CO2 atmosphere. Registration of the sensor characteristics was car-ried out by impedance spectroscopy. Interdigital sen-sors were connected into the pulse-adjustable RC-autooscillator. Electrochemical measurements were carried out in auto-oscillate regime at a frequency of quasi-resonance [11]. Microdiffraction patterns and electron microscopic images were obtained by means of transmission elec-tron microscope JEM-100CX (JEOL, Japan) (TEM) at an accelerating voltage of 100 kV. Structural analysis have been performed on the objects previously deposit-ed on a copper grid with a formvar polymer coating. Visualization of the cellular monolayer was carried out using a confocal micro-Raman spectrometer Nano-finder HE («LOTIS-TII», Tokyo, Japan).  3. RESULTS AND DISCUSSION   3.1 Oligonucleotide-MCNT LB-complex  structure  Carbon nanotubes form highly ordered LB-CNT-clusters in the LB-monolayers. According to the TEM images shown in Fig. 1a, a diameter of MCNTs in LB-cluster equals to 3 nm. Fig. 1b demonstrates that the oligonucleotide-MCNTs LB-complexes represent them-self MCNTs covered close-packed layer of oligonucleo-tide molecules, nucleotide layer thickness is of the order of 2 to 10 nm. Highly oriented MCNTs arrays are formed in the ep-itaxial growth on an iron-containing crystalline LB-films of HDTP [10]. It has been  shown that the diffrac-tion patterns of LB-nanotube clusters on the HDTP LB-films are characterized by Kikuchi lines [10] being characteristic for mono-crystallites with size of 0.5 – 1 μm. The oligonucleotide molecules located on the MCNT in LB-film are in a highly ordered state. In the zero-node of the reciprocal lattice a diffraction pattern (Laue pattern) of the LB-monolayers consisting of oligonucleo-tide-MCNT-stearic acid mixture represents itself a three reflexes with the class of symmetry C2. This indi-cates that the non-covalently functionalized by stearic acid MCNT-oligonucleotide complexes placed on HDTP LB-films are also in the monocrystal form. The oligonu-cleotide crystal interplanar spacing dp in the direction being orthogonal to the surface of MCNT is equal to 0.438 nm.  3.2 Electrochemical impedance spectroscopy  The sensors have been tested for 6 different types of coatings: 1) iron-containing HDTP LB-film and two LB-MCNT-monolayer without Ce (coating 1a) with oligonu-cleotide (coating 1b), 2) Fe-and Ce-containing HDTP LB-film and two LB-MCNT-monolayer without Ce (coating 2a) with oligonucleotide (the coating 2b), 3) Fe-containing HDTP LB-film and the two Ce-containing LB-MCNT-monolayer (coating 3a) with oligonucleotide (coating 3b).    a    b  Fig. 1 – TEM image of two-monolayer MCNTs-stearic acid mix-ture LB-films formed on Ce2(SO4)3 salt aqueous solution (а) and deposited on three HDTP LB-monolayers formed on Fe(NO3)3 salt aqueous solution with pH finished by hydrochloric acid;  (b) TEM image of two-monolayer MCNTs-oligonucleotide com-plexes-stearic acid mixture LB-film deposited on five-monolayer iron-containing HDTP LB-film  In bidistilled water, the dielectric properties of LB-MCNT-films and oligonucleotide-MCNT complexes in LB-films appear as a change of capacity of the double-charged Helmholtz layer which is formed at the inter-phase boundary between insulating barrier layer of an-odic aluminium oxide and water. At low frequency range 200 – 400 kHz a sharp decrease of capacity of the sensor placed into electrolyte has been registered, which indi-cates the presence of a significant ionic component of the double layer capacity. At the higher frequency range (600 – 1300 kHz), the capacity of sensor varies slightly due to the large distribution of relaxation times of ions (H+, OH–) in the pores and small dipole relaxation times. The measurements of electrical capacity of the sensor with deposited LB-MCNT-films showed that a change of capacity of the sensors with and without oligonucleotide has opposite sign with respect to sensor without MCNTs. The capacity change for sensors with LB-MCNT-cluster coating noncontaining oligonucleotide equals to –0.8 pF in the frequency range 500 – 900 kHz, whereas for sen-sors with LB-MCNT-oligonucleotide-clusters, the capaci-ty change equals to  +(0.8 – 1.2) pF.  CELLULAR ADHESION TO CARBON NANOTUBES- … PROC. NAP 2, 04NABM22 (2013)   04NABM22-3 Let us estimate the sensor capacity change when the ion concentration in the medium varies. According to the Maxwell-Garnett law the capacity of sensor upon its immersion in a conducting medium increases with the increase of ion concentration in the medium. The mini-mal sensor capacity is observed for 0.15 M NaCl. If the sensor were immersed in Earle's medium (pH = 7.4) that does not contain CaCl2, then its capacity increased by 65 ± 35 pF, 180 ± 20 pF and 90 ± 10 pF, that in the per-centage ratio was 6 ± 4 %, 22 ± 5 % and 10,7 ± 0,9 % of the capacity of sensor in the Earle's medium without CaCl2 for coatings type 2b, 1b and 3b, respectively. In Earle's medium with 2.5 mM CaCl2 a further increase of capacity by 33 ± 10 pF (3,3 ± 1,1 %) was observed, re-gardless of a coating type. For coatings that does not contain oligonucleotide, the capacity of sensor, which is in the Earle's medium, is higher by 235 ± 95 pF (21 ± 4 %) than in 0.15 M NaCl. For coating 1a, the capacity of sensor, which is in the Earle's medium, is higher by 66 ± 11 pF (6,0 ± 0,5 %) than in Earle's medium without CaCl2. The correspond-ing recordings for sensors with coating 2a and 3a were 117 ± 27 pF (10,7 ± 0,5 %) and 80 ± 21 pF (8,2 ± 0,5 %).  3.3 Morphology of cells in monolayer  By means of light microscopy the study of morpholog-ical characteristics of cells in a monolayer formed on the sensors on the fourth day of cell growth in culture has been performed. It is found that the number of cells on the coating 2b is less than a number of cells on the coat-ings 1b and 3b. As Fig.2 shows, morphology of cells de-pends on the coating type. On the coating 1b the popula-tion of cells is heterogeneous: there are both star-shaped cells with a high degree of flatness and having a large number of cell-cell contacts and cells being rounded and slightly flattened. On the coating 3b cells are strongly flattened and mainly elongated. Each cell has no more than three cell-cell contacts at the ends of highly elon-gated threadlike pseudopodia. Cells on the coating 2b are rounded, with a small area of contact to the surface, processes are short, cell-cell contacts are few.  3.4 Dielectric spectroscopy of cellular  monolayer  Previously it has been shown that the formation of the cellular monolayer on the sensor surface with coat-ings 1a – 3a containing no oligonucleotide is accompanied by arising of a Helmholtz double layer electric field shielding. The shielding becomes apparent as sensor ca-pacity decreasing on 1 – 3 days of cell growth and de-pends on monolayer density and a degree of intracellular communication [12]. It has been found that for sensors with coatings 1b – 3b containing oligonucleotide, the sen-sor capacity was increasing during the time of cell growth. It is stipulated by a Coulomb repulsion between negatively charged phosphate groups of oligonucleotide and negatively charged hyaluronic acid residues in cellu-lar glycocalyx, obstructing the non-antisense cell adhe-sion to oligonucleotide-contained coatings. As Fig. 3 shows, for coating 1b the sensor capacity decrement in regard to capacity of sensor without cells varies insignificantly on the second and third day of cel-lular growth as compared with the first day of growth.    a    b    c  Fig. 2 – Light microscopy images of cells grown on coating 1b (a), coating 2b (b) and coating 3b (c)  The compensation of two processes is observed, name-ly the capacity increase due to the rise of the number of cells and the capacity decrease due to the shielding effect, that is enhanced at establishing of cell-cell contacts. For the coating 2b the capacity increase on the first day of cellular growth is less by 200 pF, as compared with coat-ings 1b and 3b. This indicates that the number of cells adhered to the coating 2b is smaller than number of cells adhered to the coatings 1b and 3b. For the coatings 2b and 3b the rise of the sensor capacity on the 2nd and 3rd day of cell growth demonstrates a low rate of intracellu-lar contacts formation. These data agree with the data of cell morphology. The experimental results obtained in the work are ev-idence of the following. First, the positively charged ions Ce4+ in the HDTP LB-film of coating 2b hydrolyzed oligo-nucleotides [13]. This is accompanied by breaking of  - stacking interaction in oligonucleotide monocrystal fol-lowing formation of double helices carrying a large nega-tive charge. The negatively charged double-stranded oli-  H.V. GRUSHEVSKAYA, ET AL. PROC. NAP 2, 04NABM22 (2013)   04NABM22-4  Fig. 3 – Dependency of sensor capacity decrement on cell growth time for sensor capacity with cellular monolayer. The coatings of sensor used were coating 1b (triangle), coating 2b (square), coating 3b (rhomb)  gonucleotide prevents non-specific adhesion of negatively charged cells due to strong Coulomb repulsion. Second, oligonucleotide hydrolysis is absent for Ce3+-containing MCNT-oligonucleotide clusters of coating 3b. Therefore, - stacking interaction in the oligonucleotide monocrystal leads to the transfer of electrical charge along the - coupling chain which shields the Coulomb field of the negatively charged phosphate groups. The capacitive sensor  characteristics testify a physio-logical state of cells in different medium. The dielectric properties of the cellular monolayer depend on the transmembrane potential of cells and are determined by the state of ion channels. It was revealed that the capaci-ty of sensors with cellular monolayer depends on the composition of the measuring medium. It is shown that the capacity of sensor with cellular monolayer increases as follows: DMEM <Earle's medium (pH = 7.4) <Earle's medium (pH = 7.4) without 2.5 mM CaCl2 <0.15 M NaCl. The capacity increases by 150 ± 62 pF (4,3 ± 1,1 %) for coatings 2b and 3b, and by 300 ± 50 pF (9 ± 2 %) for coat-ing 1a when cellular monolayer is transfered from Earle's medium to Earle's medium without CaCl2. Capacity of sensors with cellular monolayer formed on the surfaces that do not contain oligonucleotide, increases by 14 ± 4 % for coatings 1a and 2a at changing the Earle's medium on the Earle's medium without CaCl2.  4. CONCLUSIONS  Thus, a nanobiosensor having an oligonucleotide-MCNT clusters layer in its sensitive coating has been developed. The sensor has a high-ordered mono-crystalline structure of LB-MCNT-clusters and a close-packed oligonucleotide layer. Such type of oligonucleo-tide deposition is promising for high-sensitive detection of antisense oligonucleotide-bonded biological objects by means of electrochemical analysis due to the dis-tance to the object must be about pair nucleotides size.  The oligonucleotide anchored in sensor coating at-tenuates cell non-antisense adhesion to sensor surface resulting in capacity increasing during cell growth. The presence of Ce in the sensor coating leads to prevent non-specific cellular adhesion.  AKNOWLEDGEMENTS  The present work was developed under the auspices of Belarusian State Program of Scientific Research «In-terdisciplinary scientific research, new incipient tech-nologies as a basis of stable innovative progress».   REFERENCES  1. T. Henning, M. Brischwein, W. Baumann R. Ehret, I. Freund, R. Kammerer, M. Lehmann, A. Schwinde, B. Wolf , Anti-Cancer Drugs 12, 21 (2001).  2. L. Ceriotti, J. Ponti, P. Colpo, E. Sabbioni, F. Rossi, Bio-sens. Bioelectron. 22, 3057 (2007).  3. J.H. 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Cellular Adhesion to Carbon Nanotubes-anchored Oligonucleotide: Electro-chemical Detection

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