Journal of Biomaterials and Nanobiotechnology, 2012, 3, 462-468 Published Online October 2012 (
Study of Photoinduced Interaction between Calf
Thymus-DNA and Bovine Serum Albumin Protein with
H2Ti3O7 Nanotubes
Rajesh Chakraborty1*, Sriparna Chatterjee2, Sandipan Sarkar3*, Pabitra Chattopadhyay1
1Department of Chemistry, Burdwan University, Burdwan, India; 2Institute of Physics, Bhubaneswar, India; 3Durgapur Institute of
Advanced Technology and Management, Durgapur, India.
Email: *, *
Received June 21st, 2012; revised July 24th, 2012; accepted August 14th, 2012
Hydrogen titanate nanotubes were synthesized by hydrothermal process using 10 M NaOH and TiO2 anatase powder.
The material synthesized was characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and
transmission electron microscopy (TEM) to ensure the structural and morphological characteristics. The interaction of
calf thymus DNA (CT-DNA) and bovine serum albumin protein with suspended aqueous solution of titanate nanotubes
was investigated using UV absorption spectroscopy and the apparent association constant was found to be, Kb= 1.68 ×
104 M
–1 and Kap=5.41 × 103 M
–1 for DNA and BSA respectively. Addition of the titanate nano material resulted
quenching of fluorescence spectra of ethidium bromide-DNA in tris HCl buffer solution and that of aqueous protein
solution. The apparent binding constant (Ksv= 5.46 × 104 M–1 for DNA binding and Ksv = 6.063 × 103 M–1 for protein
binding) was deduced from relevant fluorescence quenching data using Stern-Volmer equation.
Keywords: Photoinduced Interaction; Nanotubes; Hydrothermal Process
1. Introduction
One-dimensional nanostructures have drawn a great at-
tention due to their potential applications in a variety of
novel devices in recent past [1-5]. The most fascinating
example of such nanostructure is of course carbon nano-
tubes. Several efforts were made to synthesize nanorods
and nanowires of more complex structure and cones-
quently a number of one-dimensional nanomaterials in-
cluding metals, oxides and nitrides have also been re-
ported. Hydrothermal or solvothermal synthesis is an very
important approach to produce oxidic nanowires and
nanotubes because synthesis of these types provide ac-
cess to uniform and distinct morphologies in large scales
with remarkable reliability, selectivity, and efficiency [6].
Among the one-dimensional oxidic nanomaterials re-
ported, titanium oxide is of particular interest for its wide
applications as catalyst supports, semiconductor photo-
catalysts and sensors [7-9]. TiO2 nanoparticles are acting
as biosensors in chemical and biochemical fields and
their applications are becoming more extensive. These
probes have been applied to the ultrasensitive detection
of proteins, DNA sequencing, clinical diagnostics, etc.
TiO2 nanoparticles have also been used as carriers for
photosensitizer like porphyrins in photodynamic therapy
for cancer treatment. Titanate nanotubes of nearly 8 nm
in diameter were first reported by Kasuga and co-work-
ers, employing a hydrothermal treatment of rutile TiO2
powders in strong aqueous solution of NaOH at 110˚C
followed by HCl washing [10]. Several number of papers
were published afterwards on the structure of hydrother-
mally synthesized TiO2 which also gave birth of a num-
ber controversies. Finally Chen et al. concluded that
these nanotubes were of H2Ti3O7 structure based on dif-
fraction and high-resolution transmission electron mi-
croscopy (HRTEM) results [11].
Over the past decade a significant number of papers
have been published on the specific interaction between
photoactive nanocrystalline metal oxide semiconductor
particles and DNA or proteins or other biomolecules
[12-18]. Interestingly research works of these types open
up the possibility of the electronic transduction of DNA
sequence and hybridization, as well as development of
new electrochemical probes for DNA-binding proteins,
both of which are major challenges in bioelectronics [16,
19,20]. Deoxyribonucleic acid (DNA) is unique genetic
molecule in any living organism. Consequently the
qualitative and quantitative analysis of nucleic acids is
*Corresponding authors.
Copyright © 2012 SciRes. JBNB
Study of Photoinduced Interaction between Calf Thymus-DNA and
Bovine Serum Albumin Protein with H2Ti3O7 Nanotubes 463
attracting a great attention because this is the material
base of genetic inheritance. On the other hand Serum
albumin is the most plentiful protein in blood plasma. It
is a large globular protein containing 582 amino-acid
residues with a molecular weight of 69,000 Dalton and
two tryptophan moieties at positions 134 and 212 as well
as tyrosine and phenylalanine [21] Each protein molecule
can carry seven fatty acid molecules. They bind in deep
crevices in the protein, burying their carbon-rich tails
safely away from the surrounding water. Serum albumin
also binds to many other water-insoluble molecules. The
strong affinity of BSA to the water molecules has made
the protein an automatic choice for the interaction studies
in the present work. Moreover BSA is used as protein
model because of its stability, its lack of effect in many
biochemical reactions, and its low cost. BSA has numer-
ous biochemical applications including Enzyme-Linked
Immunosorbent Assay immunoblots, and immunohisto-
chemistry [22]. It is also used as a nutrient in cell and
microbial culture. BSA is used to stabilize some enzymes
during digestion of DNA and to prevent adhesion of the
enzyme to reaction tubes. This protein does not affect
other enzymes that do not need it for stabilization. The
binding properties of BSA with various drugs have been
fully investigated by many researchers [23-25]. In this
present work, interaction between suspended H2Ti3O7
nanotubes (in aqueous solution) and CT-DNA, followed
by BSA protein and was examined by UV and fluores-
cence spectroscopy. As the natural fluorescence intensity
of DNA is very weak, ethidium bromide (EB) was used
to enhance the fluorescence intensity while BSA shows
intrinsic fluorescence due to the presence of aromatic
amino-acid residues.
2. Materials and Methods
2.1. Materials
CT-DNA and TiO2 (AR grade) was purchased from
Merck (Mumbai, India) while ehidium bromide and bo-
vine serum albumin protein was purchased from Sigma
Aldrich. Approximately 10–4 M stock solution of CT-
DNA was prepared by dissolving proper amount of DNA
in 1 M tris buffer.
2.2. Preparation of\Hydrogen Titanate
Nanotubes (HTO)
A 1.0 g potion of anatase TiO2 powder was dissolved in
10M NaOH solution and the resulting suspension was
then transferred in an autoclavable reaction chamber. The
reaction chamber was autoclaved in an oven at a tem-
perature of 130˚C for 22 hours. The product was filtered
after cooling down to room temperature and thoroughly
washed with 1 M HCl and water to remove any trace of
NaCl produced during washing [26]. Subsequently the
material was dried at ambient temperature.
2.3. Characterization of HTO
The powder X-ray diffraction (XRD) data were recorded
from a PANalytical X’pert Pro diffractometer with Cu
Kα radiation. The morphology of the nanostructured
samples was studied with use of a Jeol JSM-840 scan-
ning electron microscope (SEM) operating at 120 keV.
TEM studies of nanotubes were carried out with a FEI
Tecnai TEM equipped with a LaB6 filament and operated
at 200 kV. TEM samples were prepared by placing a
drop of the ultrasonically dispersed powder (in ethanol)
on a carbon-coated copper grid and drying in air.
2.4. Fluorescence Quenching Measurements
A Hitachi F-4500 fluorescence spectrophotometer was
used to perform the fluorescence quenching measure-
ments. The excitation wave length of EB-DNA was kept
fixed at 522.0 nm while the emission wave length was
chosen at 400.0 nm. The excitation and emission slit
length (5 nm and 10 nm respectively) wave lengths as
well as the scan speed (2400 nm/min) were kept fixed
throughout the experiment. For BSA-HTO interaction
study the excitation and emission wavelength was kept
fixed at 510 nm and 435 nm respectively with all the
other conditions remaining the same as before.
2.5. Absorption Spectral Measurements
To carry out the absorption spectral measurements, a
JASCO V-570 UV-Vis spectrophotometer was used. For
each measurement, 4.0 mL solution, containing DNA,
suspended solution of nanoparticle and tris buffer was
taken. At first the UV-vis spectrum was recorded for a
solution without DNA. For the rest of the solutions, DNA
was added with an increment of 0.05 mL each and then
the spectra for all the solutions were recorded in the
range of 200 - 800 nm. BSA absorbs light energy around
280 nm. So the absorption spectra for purely aqueous
solution of BSA (10–2 M) was recorded at the beginning
and then suspended aqueous solution of HTO was added.
3. Results and Discussions
3.1. Structural and Morphological Studies of
X-ray diffraction pattern of HTO is shown in Figure 1
This diffraction pattern mostly matches with the mono-
clinic crystal structure of H2Ti3O7 (space group C2/m
(12)) (JCPDS Card no. 41-0192). Figure 2 shows the
Copyright © 2012 SciRes. JBNB
Study of Photoinduced Interaction between Calf Thymus-DNA and
Bovine Serum Albumin Protein with H2Ti3O7 Nanotubes
Figure 1. XRD spectra of HTO.
Figure 2. SEM image of H2Ti3O7 nanotubes.
SEM images of the nanostructured samples hydrother-
mally synthesized from anatase powder. The scanning
electron micrograph shows the formation of one dimen-
sional (1-D) nanostructures. Further TEM study (Figure
3) clearly shows that the each 1-D units are actually hol-
low, multiwalled nanotubes with inner diameter of 4 - 5
nm and the outer diameter of 9 - 10 nm.
3.2. UV Absorption Study by CT DNA and BSA
with HTO
Figure 4 shows the absorption spectra of CT-DNA in
presence of suspended aqueous solution of HTO. The
concentration of suspended HTO was kept constant for
all sets of experiments while the concentration of DNA
was increased uniformly. In presence of HTO the ab-
sorbance of DNA increases considerably with increasing
concentration of DNA. Similar type of experiment was
Figure 3. TEM image of H2Ti3O7 nanotubes.
Figure 4. Electronic spectral titration HTO with CT-DNA
at 266 nm in tris-HCl buffer; [HTO] = 2.5 × 10–5; [DNA]: (a)
0.0, (b) 6.2 × 10–6, (c) 10.0 × 10–6, (d) 15.2 × 10–6, (e) 20.6 ×
10–6 mol·L–1. Arrow indicates the direction of change upon
the increase of DNA concentration.
performed with BSA and HTO and the only difference is
that the concentration of BSA was kept constant through-
out the experiment. Figure 5 shows the gradual increase
of absorption spectra with increasing concentration of
HTO. To make it confirm that the absorption change
which we observed in the spectrum was not due the ex-
perimental error, baseline corrections were done for all
the measurements. Therefore the results clearly indicate
that there is a interaction between suspended HTO and
CT DNA/BSA through the formation of some complex
of the type HTO7DNA, HTO-BSA [27,28].
3.3. Fluorescence Quenching Study of EB-CT
As it has been mentioned earlier that the natural fluores-
Copyright © 2012 SciRes. JBNB
Study of Photoinduced Interaction between Calf Thymus-DNA and
Bovine Serum Albumin Protein with H2Ti3O7 Nanotubes 465
cence intensity of DNA is very weak, ethidium bromide
(EB) was used as fluorescence probe for DNA. Figure 6
shows that addition of suspended aqueous solution of
HTO to a solution of EB-DNA resulted in a decrease in
fluorescence emission intensity of EB-DNA. HTO solu-
tion was added in increasing concentration keeping the
EB-DNA concentration constant for all the measure-
ments. Fluorescence quenching study of BSA was car-
ried out in similar fashion keeping the concentration of
BSA solution fixed. Figure 7 shows the quenching of
Figure 5. Electronic spectral titration of HTO with BSA at
280 nm in water.[BSA] is constant and [HTO]: (a) 1.25 ×
105; (b) 2.5 × 105; (c) 3.75 × 105; (d) 5.0 × 105; (e) 6.25 ×
105 mol·L1. Arrow indicates the direction of change upon
the increase of HTO concentration.
Figure 6. Emission spectra of the CT-DNA-EB system in
tris–HCl buffer upon the titration of the HTO. kex = 522 nm;
[EB] = 9.6 × 10–5 mol·L–1; [DNA] = 1.25 × 10–5; [Complex]:
(a) 0.0; (b) 1.25 × 10–5; (c) 2.5 × 10–5; (d) 3.75 × 10–5; (e) 5.0
× 10–5 mol·L–1. Arrow shows the intensity change upon the
increase of the complex concentration.
Figure 7. Emission spectra of the aqueous solution of BSA
upon the titration of the HTO. [BSA] is constant [HTO]: (a)
1.25 × 105; (b) 2.5 × 105; (c) 3.75 × 105; (d) 5.0 × 105; (e)
6.25 × 105 mol·L1. Arrow indicates the direction of change
upon the increase of HTO concentration.
BSA protein with increasing concentration of HTO.
Baseline correction was done for all the measurements to
get rid of the probable scattering effect due to the sus-
pended nature of HTO in aqueous medium. It ensures
that fluorescence quenching occurs only due to interact-
tion between CT DNA/BSA and HTO.
3.4. Determination of Binding Constant between
In order to further illustrate the binding strength of the
HTO with CT-DNA, the intrinsic binding constant Kb
was determined from the spectral titration data using the
following equation:
where [DNA] is the concentration of DNA,
a and
correspond to the extinction coefficient, respectively, for
the free HTO, for each addition of DNA to the suspended
solution of HTO and for the HTO-DNA complex in the
fully bound form. A plot of
[DNA], gives Kb, the intrinsic binding constant as the ratio
of slope to the intercept. From the
versus[DNA] plot (Figure 8), the binding constant Kb for
the HTO-DNA complex was determined to be 1.68 × 104
M–1 (R = 0.97208 for four points).
Fluorescence intensity of EB bound to DNA at 522 nm
shows a decreasing trend with the increasing concentra-
tion of the HTO (Figure 6). The quenching of EB bound
to DNA by the titanate nanorods are in agreement with
Copyright © 2012 SciRes. JBNB
Study of Photoinduced Interaction between Calf Thymus-DNA and
Bovine Serum Albumin Protein with H2Ti3O7 Nanotubes
the linear Stern-Volmer equation:
II1K Q
where I0 and I represent the fluorescence intensities in
absence and presence of quencher, respectively. Ksv is a
linear Stern-Volmer quenching constant, Q is the con-
centration of quencher. In the quenching plot (Figure 9)
of I0/I versus [complex], Ksv value is given by the ratio of
the slope to intercept. The Ksv value for the complex is
5.46 × 104 (R = 0.96771 for four points), suggesting a
strong affinity of the titanate nanorods to CT-DNA. Again
the apparent binding constant (Kap) due to interaction
between HTO with BSA can be determined following the
obs0c0ap c0
1AA1AA 1KAAHTO 
[DNA]/ (ε
- ε
Figure 8. Plot of [DNA]/(
f) vs [DNA] for the absorp-
tion titration of CT-DNA with the HTO in Tris-HCl buffer.
1.05 1.10 1.15 1.20 1.25 1.30
I0/ I
Figure 9. Plot of I0/I vs [HTO] for the titration of suspended
aqueous solution of [HTO] to CT-DNA-EB system.
where A0 and Ac are the absorbance of BSA and complex
respectively at 280 nm and Aobs is the observed absor-
bance of the solution containing different concentrations
of suspended HTO at 280 nm. From the plot of log[A0
A]/A vs 1/[HTO] (Figure 10) for the absorption titration
of BSA with the HTO the Kap was determined to be 5.41
× 103 (R = 0.9558 for four points). Fluorescence intensity
of BSA at 280 nm shows the similar trend as the DNA
which is mentioned earlier and the binding constant can
be determined by Stern-Volmer equation as usual. In the
quenching plot (Figure 11) of I0/I versus [HTO] for
BSA-HTO interaction, Ksv value is given by the ratio of
the slope to intercept. The Ksv value for the complex is
6.063 × 103 (R = 0.9953 for four points). The binding
constant values for both cases (Kap and Ksv) indicate a
considerably strong affinity of the HTO to BSA.
20000 30000 4000050000 60000 70000 80000
Figure 10. Plot of log[A0 – A]/A vs 1/ [HTO] for the absorp-
tion titration of BSA with the HTO in aqueous medium.
Figure 11. Plot of I0/I vs [HTO] for the titration of sus-
pended aqueous solution of HTO to BSA.
Copyright © 2012 SciRes. JBNB
Study of Photoinduced Interaction between Calf Thymus-DNA and
Bovine Serum Albumin Protein with H2Ti3O7 Nanotubes 467
4. Conclusion
Study of photoinduced interaction between HTO and CT
DNA using UV and fluorescence spectroscopic tools
established that the interaction is quite strong which is
also revealed by the high binding constant. The present
work may be helpful in understanding the interaction
between various photosensitizers and DNA. Clinical ap-
plication of the existing photosensitizer is associated with
some problems such as aggregation of photosensitizers in
aqueous medium, decrease in quantum yield due to ag-
gregation, toxicity etc. HTO nano particles can overcome
all the above mentioned problems. This observation can
also be considered with importance to investigate such
interaction between nanotubes and DNA for future ap-
plication in photodynamic therapy. This study of interac-
tion of HTO with physiologically important BSA protein
will provide some necessary information for the design
and application of new drugs. ultrasensitive detection of
proteins, clinical diagnostics etc.
5. Acknowledgements
Financial assistance from UGC-DAE Center for Scien-
tific Research, Kolkata is gratefully acknowledged. The
authors are obliged to Prof. P. Ayyub of Department of
Condensed Matter Physics and Material Sciences, Tata
Institute of Fundamental Research for providing TEM
and SEM facilities.
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