Study of Photoinduced Interaction between Calf Thymus-DNA and Bovine Serum Albumin Protein with H2Ti3O7 Nanotubes

doi:10.4236/jbnb.2012.34047

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Journal of Biomaterials and Nanobiotechnology, 2012, 3, 462-468

http://dx.doi.org/10.4236/jbnb.2012.34047 Published Online October 2012 (http://www.SciRP.org/journal/jbnb)

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: *[email protected], *[email protected]

Received June 21st, 2012; revised July 24th, 2012; accepted August 14th, 2012

ABSTRACT

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.

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

HTO

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

Study of Photoinduced Interaction between Calf Thymus-DNA and

Bovine Serum Albumin Protein with H2Ti3O7 Nanotubes

464

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

DNA by HTO

As it has been mentioned earlier that the natural fluores-

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 ×

10−5; (b) 2.5 × 10−5; (c) 3.75 × 10−5; (d) 5.0 × 10−5; (e) 6.25 ×

10−5 mol·L−1. 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 × 10−5; (b) 2.5 × 10−5; (c) 3.75 × 10−5; (d) 5.0 × 10−5; (e)

6.25 × 10−5 mol·L−1. 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

CT DNA and HTO

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:



















afbfbbf

DNADNA1 K













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



 versus

[DNA], gives Kb, the intrinsic binding constant as the ratio

of slope to the intercept. From the









DNA





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

Study of Photoinduced Interaction between Calf Thymus-DNA and

Bovine Serum Albumin Protein with H2Ti3O7 Nanotubes

466

the linear Stern-Volmer equation:





0sy

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

equation:















obs0c0ap c0

1AA1AA 1KAAHTO 

6.0×10

–

8.0×10

–

1.0×10

–

1.2×10

–

1.4×10

–

1.6×10

–

1.8×10

–

2.0×10

–

2.2×10

–

[DNA]

7.2×10

–

7.0×10

–9

6.8×10

–9

6.6×10

–9

6.4×10

–9

6.2×10

–9

6.0×10

–9

5.8×10

–9

[DNA]/ (ε

- ε

)

Figure 8. Plot of [DNA]/(



a −



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

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

I0/ I

[HTO]

Io/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

log

[A0

-A]/A

1/[HTO]

Iog[AO-A]/A

Figure 10. Plot of log[A0 – A]/A vs 1/ [HTO] for the absorp-

tion titration of BSA with the HTO in aqueous medium.

2.0x10

-5

3.0x10

-5

4.0x10

-5

5.0x10

-5

6.0x10

-5

1.05

1.10

1.15

1.20

1.25

1.30

[

HTO

]

[HTO]

Figure 11. Plot of I0/I vs [HTO] for the titration of sus-

pended aqueous solution of HTO to BSA.

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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