Materials Sciences and Applicatio n, 2011, 2, 940-945
doi:10.4236/msa.2011.27125 Published Online July 2011 (http://www.SciRP.org/journal/msa)
The Physical and Biological Properties of
NanoTiO2 Material
Xiao Feng Pang
Institute of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu; P.R.China and In-
ternational Centre of Materials Physics, Chinese Academy of Sciences, Shengyang, P.R.China.
Received May 28th, 2010; revised June 12th, 2010; accepted May 30th, 2011.
ABSTRACT
The physical and biological properties of TiO2 materials including NanoTiO2, micrometerTiO2 and NanoTiO2 tubes have
been studied using scanning electron and infrared spectrometer, X-ray diffraction instrument as well as 3-(4,5- di-
methylthiazol 2-yl)-2,5 diphenyltetrazo-lium bromide (MTT) colorimetric method, respectively. These materials are pre-
pared by chemical deposition and anode oxidation methods, respectively. The sizes of NanoTiO2 are 80 nm and 1000 nm,
respectively, their infra red properties of absorption are differen t, the characteristic peaks of the former are 1271, 1615,
2957 and 3422 cm–1, the latter are 1645 and 2356 cm–1. The NanoTiO2 tubes can be formed by anode oxidation method,
its diameters are between 50 - 100 nm, different NanoTiO2 tubes contain different components of oxygen and titanium. In
MTT experiment we discover the changes of properties of proliferation of the liver and chick embryo fibroblast cells un-
der influences of NanoTiO2 relative to those of the controlled groups, when small NanoTiO2 suspension is added in these
cultivated liquids of cell, but the influence of NanoTiO2 on the proliferation of the person’s liver cell is still very small,
therefore, the toxicities of N anoTi O 2 co nt ai ni ng 80 nm and 1000 nm to these cells are still first score.
Keywords: NanoTiO2, Micrometer TiO2, NanTiO2 Tube, Infrared and X-Diffraction Spectrum, MTT Method, Cell,
Biological and Physical Property
1. Introduction
At present, the Nanomaterials and Nanotechnology and
Nanoinstrument have been greatly developed and applied
extensively in the industry, agriculture, science and
medicine as well as our living. A lot of Nanomaterials
have been made in bulk in factory and were sold in mar-
kets. In such a case it is very necessary to know whether
these Nanomaterials are safe, or speaking, have toxicity
to the person’s and animal’s health [1-4]. This problem
attracts scientist’s and government’ attentions in the
world. Thus studying the safety or toxicity of the Nano-
materials has important significances in sciences and
applications. In this paper we will study the physical and
biological properties of NanoTiO2.
TiO2 is a kind of crystal, in which one titanium (Ti)
atom combines with six oxygen (O) atoms, one oxygen
atom links again three titanium atoms to form an octagon.
Thus, TiO2 belongs in the inclined crystalline system and
is a semiconductor material. In this structure of crystal
the width of forbidden band between the valence band
and conductive band is between 3.0 eV - 3.2 eV. There-
fore, TiO2 can easily absorb the ultraviolet light with
wavelength of 387 nm and414nm. Just so, single crystal
TiO2 can decompose water and other substances under
action of the light. Hence TiO2 is a good material of
photocatalytic materials. Its photocatalytic theorem can
be described as follows. Under action of incident light
the electrons in the valence band transit into the conduc-
tive band (e–), then the holes (h+) occur in valence band
in TiO2. This reaction is represented by


–+
22
TiO+ hνTiOe ,h. These electrons and holes
occurred are shifted on the surface of TiO2 under action
of an electric-field and can interact further with other
materials through the following oxidation and reduction
reactions:
++
2
h+H OH+H
+
h+OH OH
, ––
22
O+e O
–
22
O+H OOOOH+OH, ,
22
2 OOOHO+HO 2
––
222
OOOH+H O+eH O+OH,
––
22
H O+eOH+OH,.

The Physical and Biological Properties of NanoTiO Material941
2
In these processes a great number of free radicals
of ,and are generated, which have
strong ability of oxidation and can interact and react with
plenty of materials, such as chloroform, PCBS, organic
compounds, formaldehyde. Therefore, the NanoTiO2
have extensively applications in medicine, agriculture
and industry, containing chemical engineering, paint,
paper, plastic, rubber, chemical fiber, electric-appliances,
cosmetics and food packaging [4-7].
-
OH -
2
OOOOH
For the NanoTiO2, which is different from that of bulk
TiO2due to its scale to become very small, such as, it has
larger rate of surface area versus volume about 70 m2/g,
its surface energy raises, the ratio of number of atom
between the surface and interior increases, the number of
the coordination of atoms in it is lowered, when com-
pared with that in bulk [3-4]. Thus the physical and
chemical activities as well as unstablities of NanoTiO2
are increased correspondingly. Meanwhile, it is also quite
difficultly resolved in water, but when a lot of NanoTiO2
are collected together, they can combines and concretes
each other to form gluey state in water. These gluey
states of the NanoTiO2 are easily spitted, if some elec-
trolytes are added into this solution, or the PH value of
solution is changed. Meanwhile, it can also form some
new bases or chemical compound, such as, TiOH, K2O-6
TiO2 or K2Ti6O13, etc., with water molecules or ions,
involving OH, NH2, COOH, C = O, through attraction
interaction between the particles with charges on the
surface. Therefore, it is very necessary to investigate
in-depth the physical and biological properties of Nano-
TiO2.
2. Experimental Method
We prepare three kinds of TiO2 materials containing the
NanoscaleTiO2, microscale TiO2 and NanoTiO2 tubes by
using chemical deposition and anode oxidation methods,
respectively. Their features of structures are measured
using Scanning Electron Spectrometer (SEM), respec-
tively, its infrared properties of absorption and
X-diffraction spectra are measured using a Nicolet Nexus
670-FT-IR spectrometer with resolution of 4 cm–1 and
X-ray diffraction spectrometer, respectively. The prolif-
eration and toxicity of NanoTiO2 to the person’s liver
and chick embryo fibroblast (CEF) cells are determined
by MTT colorimetric method [8].
So-called MTT colorimetric method [8] is just a very
effective way checking the states of activity and prolif-
eration of the cells. In this way the coloration substance
used is MTT. The MTT is an abbreviation of 3-(4,
5-dimethylthiazol 2-yl)-2, 5 diphenyltetrazolium bromide,
which is a sort of dye accepting hydrogen atom. In the
mitochondrion of cell, the externally applied yellow te-
trazolium salt 3-(4, 5-dimethylthiazol 2-yl)-2, 5 di-
phenyl-tetrazolium bromide (MTT, Amersco) will be
reduced and become further, under the action of dehy-
drogenase of amber acid, as a blue insoluble formazan
form, which is eventually deposited in the cell after this
reaction. But the dead cell has not this function and effect.
The dimethylsulfoxide (DMSO) added can resolve the
blue insoluble formazan. The quantity of the blue insolu-
ble formazan produced after the resolution is propor-
tional with the number of cell participated in this process.
Thus we can determine indirectly the number of the cells
through measuring the strength of absorption of the light
with determinate wavelengths in this case. The strength
of light can be measured and collected by enzymic im-
munoassay instrument and spectrophotometer. Then we
can determine the number of proliferation and activity of
cells or of biological factors in the cells, thus we can as-
sess the safety or toxicity of NanoTiO2 to the cell, etc.,
according to the toxicology. The advantages of the me-
thod measuring this security or toxicity is fast and accu-
rate, and have higher sensitivity and very good repeat-
ability. Therefore, we here utilize this method to assess
the influences of NanoTiO2 on the proliferation states of
person’s liver cell (L-20) after it interacts with the Nano-
TiO2 by traditional biological technique. The experimen-
tal process in this method is as follows.
2.1. Cell Growth
The person’s liver and primary chick embryo fibroblast
(CEF) cells are prepared, their secondary cultures are
grown in the 5% CO2 enriched incubator with tempera-
ture of 37˚C. The person’s liver and CEF cells were
grown in RPMI1 640(Hyclone,American) supplemented
with 5% fetal calf serum (FCS, Biological Industries,
BaiAn, China). Microscopic inspection to them verifies
that the cells are not contaminated from third passage,
and so forth.
2.2. The Nanotio2 is Added into the Group
Solutions
In our experiments, 4 × 105 cells per well are seeded in
60-well micro-culture plates and allowed to continually
grow. The 60-well cells are added into the 100 μL/well
foster liquids containing the fetal calf serum in which the
1 mL/250 mL insulin liquid is included. The NanoTiO2s
are added into these wells to study the influences of the
NanoTiO2 on the proliferation behavior of the person’s
liver and CEF cells. In this experiment these cells are
separated as controlled and experimental groups, which
are all 30 well. The NanoTiO2s are assigned in the fol-
lowing rule. (a) the first or controlled group is 30 wells,
which the 15 mL/ well foster liquid without the fetal calf
serum is added into; (b) the second or experiment group
1 has 10 wells, in which the 5 μL NanoTiO2 suspension

The Physical and Biological Properties of NanoTiO Material 942 2
and 10 μL foster liquid without the fetal calf serum are
added, the concentration of NanoTiO2 achieves 43
μg/mL; (c) the third or experiment group 2 contains 10
wells, in which there is 10 μL NanoTiO2 suspension and
5uL foster without fetal calf serum, the concentration of
the NanoTiO2 achieves 86 ug/mL; (d) the fourth or ex-
periment group 3 contains 10 wells, in which 15 uL
NanoTiO2 suspension is added, the concentration of the
NanoTiO2 achieves 129 μg/mL. The above four groups
are all placed into the CO2 enriched incubator with 37˚C
to develop about 24 hours. The cell proliferation was
evaluated after 24 h.
2.3. Measurement of the Proliferation of the
Person’s Liver Cell
We observe and measure the proliferation of the person’s
liver cell (L-20) in above conditions by the MTT method
[8-10] and calculate the proliferation rate of cell by using
these experimental data.. In the calculation we should
firstly measure the increased values of mitochondrial
dehydrogenase activity as the number of cells increases.
In such a case, 100 uL/ well MTT solution is prepared in
PBS (5 mg/mL) and further diluted (10%v/v) in RPMI
1640. The cell growth medium is aspirated. In this case
the 100 μL of MTT solution are added into each well in
the above four groups. The cells are then further incu-
bated for 4 h at 37˚C Excess of MTT solution is removed,
after this the 100 μL/well DMSO is added into dissolve
the blue crystals formed in the cells. The absorption
strength of the DMSO solution and values of optical
density (OD) of each well to the incident light with wa-
velength of 570 nm for the controlled and experimental
groups are measured spectrophotometrically by DG3022
enzymicimmunoassay instrument, respectively. Finally
we can find out the cell proliferation rate (CPR) for the
person’s liver cell by using the experimental data and the
formula: CPR = [(Fexp – Fcon)/Fcon] × 100%, here Fexp is
the value of optical density of experimental group, Fcon is
the value of optical density of controlled group.
3. Experimental Results and Discussion
3.1. Properties of Microscale TiO2 and NanoTiO2
Tubes
The structures of two kind of TiO2 using SEM are shown
in Figure 1(a) and (b), respectively. This figure shows
that the NanoTiO2 is 80nm, other is 1000nm. We collect
their spectra of infrared absorption by 670 FT-IR instru-
ment, which is shown in Figure 2. We see from this fig-
ure that their infrared absorptions are different not only
the strengths and frequencies of peaks of absorption but
also the amounts of peaks, the new peaks at 2422, 2956
and 1271 cm–1 occur in the NanoTiO2, they are the char-
(a)
(b)
Figure 1. The images of SEM of TiO2 powder. (a)The image
of NanoTiO2 powder; (b) The image of micrometerTiO2
powder.
materal 2
2356
1271
1645
materal 1
2957
3422
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Absorbance
1000 2000 3000
cm–1
Figure 2. The spectra of infrared absorption for NanoTiO2
(material1) and micro TiO2 (material 2).
acteristic peaks of NanoTiO2. However, in micrometer
TiO2 there is only a small new peak at 2355 cm–1. Only
the peak at 1645 cm–1 is same for The NanoTiO2 and

The Physical and Biological Properties of NanoTiO2 Material 943
micrometerTiO2. Obviously, this is due to the changes of
structure and sizes of molecules of TiO2, the new peaks
in Figure 2 indicate that there are many new radicals or
base groups in NanoTiO2. This is just the feature of Na-
nomaterials.
and titanium are shown in Figure 5, where Figure 5a is
the result obtained from solution of phosphoric acid and
hydrofluoric acid in which the weight ratio of oxygen
and titanium are 17.54% and 82.46%, respectively, their
volume ratios are 38.91% and 61.09%, respectively,
Figure 5b is the result obtained from solution of hydro-
fluoric acid in which the weight ratio of oxygen and
titanium are 24.22% and 75.78%, respectively, their
volume ratios are 48.90% and 51.10%, respectively.
These results manifest that not only different NanoTiO2
tubes can be obtained by different conditions in the an-
ode oxidation but also the products of NanoTiO2 tubes
manufactured by this method are very pure and contain
not impurities.
We also use SEM to measure the feature and size of
NanoTiO2 tubes formed by anode oxidation method,
which are shown in Figures 3-4, which are born from the
surface of titanium alloy in the solutions of hydrofluoric
acid as well as phosphoric acid and hydrofluoric acid,
respectively. From these figures we see clearly the oc-
currences of a great number of Nanotubes, which are
distributed densely and nonuniformly on the surface,
their sizes of diameter are different and between 50 nm
and 100 nm. On the other hand, we determine the solubility of the
NanoTiO2 in some liquids . Experiments show that the
NanoTiO2 is insoluble in the unorganic ( as D-Han’k
liquid) and organic (as DMSO) solutions and alcohols
(95%) and t1 640 foster liquid without fetal calf serum,
etc.
In the meanwhile, we measure the X-diffraction spec-
trum of NanoTiO2 tubes using X-ray diffraction instru-
ment. These results for different components of oxygen
3.2. The Biological Properties of the NanoTiO2
We measured the values of optical density (OD) in
MTT experiment of liver cell and changes of prolifera-
tion of CEF by MTT method [8].
a) The OD values for the controlled group (30 wells)
obtained are as follows,
0.37, 0.31, 0.41, 0.46, 0.54, 0.45, 0.44, 0.45, 0.42,
0.43, 0.50, 0.43, 0.51, 0.54, 0.41, 0.39, 0.52, 0.47, 0.46,
o.31, 0.54, 0.38, 0.42, 0.47, 0.34, 0.41, 0.42, 0.44, 0.46.
Therefore, the average value of the OD of the con-
trolled group is 0.43 + 0.05
b) The OD values for the experimental groups are as
follows. The OD values of the experimental group 1 (10
wells) are 0.41, 0.44, 0.41, 0.62, 0.35, 0.42, 0.77, 0.49,
0.54, 0.45. Thus, its average value of OD is 0.49 + 0.09,
its PRC value is +13.95%.
Figure 3. The images of SEM of NanoTiO2 tube formed in
the solution of hydrofluoric acid.
The OD values of the experimental group 2 (10 wells)
are 0.44, 0.59, 0.40, 0.56, 0.40, 0.46, 0.32, 0.20, 0.43,
0.35. Then its average value of OD is 0.42 + 0.08, its
PRC value is –2.32%.
The OD values of the experimental group 3 (10 wells)
are 0.45, 0.44, 0.42, 0.46, 0.46, 0.40, 0.45, 0.58, 0.35,
0.32.Then its average value of OD is 0.43 + 0.05, its PRC
value is 0.0%.
3.3. Discussion of the Results
From above results we can find out the average value of
OD for the experimental group which is 0.45 + 0.07, its
CPR value is +0.02%. This shows that the NanoTiO2 in
fluences not basically the proliferation of the person’s liver
cell (L-20).
Figure 4. The images of SEM of NanoTiO2 tube formed in
the solution of phosphoric acid and hydrofluoric ac id.

The Physical and Biological Properties of NanoTiO Material 944 2
(a)
(b)
Figure 5. X-diffraction spectra of NanoTiO2 tubes with different components of oxygen and titanium in different conditions.
In order to verify above result we further study and
measure the changes of the proliferation behavior of the
chick embryo fibroblast (CEF) cell by above MTT me-
thod [8-10] , when the NanoTiO2 of 80 nm and the TiO2
of 1000 nm are added into these cells, respectively. We
obtain their OD values and find correspondingly out the
relative cell proliferation rate (RCPR) of chick embryo
fibroblast by the formula, (Fexp/Fcon) × 100%. The OD
values and corresponding RCPR values and evaluation of
their toxicity scores in the cases of interaction times of
24 hours and 48 hours are shown in table 1 and 2 for the
six samples in the two groups, respectively, where 100%
group denotes this case in which the NanoTiO2 samples
are not added into the water, the 50% group denotes that
the 50% water is added into the sample, 25% group de-
notes that 75% water is added into the sample. From the
table 1 and 2 we see that the degrees of toxicity for the
chick embryo fibroblast cell are all first score. This
shows that the toxicities of both the NanoTiO2 and TiO2
of 1000 nm to the cells are all lower. Therefore, the Na-
noTiO2 is safe in the living systems.
4. Conclusions
In this paper we investigated the physical and biological
properties of TiO2 materials including NanoTiO2, microme-
terTiO2 and NanoTiO2 tubes using scanning electron and
infrared spectrometer and X-ray diffraction instrument as
well as MTT colorimetric method, respectively.

The Physical and Biological Properties of NanoTiO Material945
2
Table 1. The OD values, Relative cell proliferation Rate
(RCPR) and toxicity Score of CEF cell after 24 hours.
Groups 24hours
groups OD value(X+S)RCPR (%) Score
100% T1 0.298±0.006, 98.3 1
50% T1 0.298±0.006, 98.8 1
25% T1 0.2995±0.007 99 1
100% T2 0.276±0.007 91.4 1
50% T2 0.286±0.003 94.7 1
25% T2 0.294±0.009 97.4 1
Control
group 0.302±0.009 100 0
Table 2. The OD values, Relative cell proliferation Rate
(RCPR) and toxicity Score of CEF cell after 48 hours.
Groups 48hours
groups OD value(X+S)RCPR (%) Score
100% T1 0.309±0.01 90.6 1
50% T1 0.320±0.003 93.8 1
25% T1 0.327±0.008 95.9 1
100% T2 0.307±0.007 90.0 1
50% T2 0.319±0.003 93.5 1
25% T2 0.325±0.008 995.3 1
Control
gronp 0.341±0.009 100 0
These materials used are prepared by chemical deposi-
tion and anode oxidation methods, respectively. The siz-
es of NanoTiO2 are checked using scanning electron
spectrometer and 80nm and 1000 nm, respectively, di-
ameters of the NanoTiO2 tubes are between 50 - 100 nm.
The infrared properties of absorption for the NanoTiO2
and micrometerTiO2 are different, the characteristic
peaks of the former are 1271 cm–1, 1615 cm–1, 2957 cm–1
and 3422 cm–1, the latter are 1645 cm–1 and 2356 cm–1.
Meanwhile, we can obtain different components of oxy-
gen and titanium in the NanoTiO2 tubes in different con-
ditions by the anode oxidation method. In MTT experi-
ment we discover the changes of properties of prolifera-
tion of the liver and chick embryo fibroblast cells under
influences of NanoTiO2 relative to those of the controlled
groups, when small NanoTiO2 suspension is added in
these cultivated liquids of cell, but the influence of Na-
noTiO2 on the proliferation of the person’s liver cell is
still very small, therefore, the toxicities of NanoTiO2
containing 80 nm and 1000 nm to these cells are still first
score.
5. Acknowledgements
The authors would like to acknowledge the National
“973” project of China for financial support (grate No:
2007CB936103)
REFERENCES
[1] R. F. Service, “Nanomaterials Show Signs of Toxicity,”
Science, Vol. 300, No. 11, November, 2003, pp. 243-247.
doi:10.1126/science.300.5617.243a
[2] D. C. Wu and C. L. Du, “Noano Fibers,” Chemical In-
dustry Press, Beijing, 2003.
[3] K. Sunada, Y. Kikuchi, K. Hashimolo and A. Fujishima,
“Bactericidal and Detoxification Effects of TiO2 Thin
Film Photocatalysts,” Environmental Science and Tech-
nology, Vol. 32, No. 5, May, 1998, pp. 726-730.
doi:10.1021/es970860o
[4] X. F. Pang, “Biopysics,” Press of University of Electronic
Science Technology of China, Chengdu, 2007.
[5] S. Daye and Y. L. Guo, “Cell Singal Transmition,” Sci-
ence Press, Bejing, 2001.
[6] R. N. Jorissen, F. Walker and N. Pouliot, et al., “Epider-
mal Growth Factor Receptor Mechanisms of Activation
and Signaling,” Experimental Cell Research, Vol. 284,
No. 1, January, 2003, pp. 31-53.
doi:10.1016/S0014-4827(02)00098-8
[7] W. Han, Y. D. Wang and Y. F. Zheng, “In Vitro Bio-
compatibility Study of NanoTiO2 Materials,” Advanced
Materials Research, Vol. 47-50, October, 2008, pp. 1438-
1441. doi:10.4028/www.scientific.net/AMR.47-50.1438
[8] Z. Q. Situ and J. Z. Wu, “Cell Culture,” World Press,
Xi’an, 1996.
[9] M. H. Zhang, S. W. Ding, Z. X. Wang and Y. Z. Zhang,
“Synthesis of Mesoporous NanoTiO2 Doped with sn by
Auto-Assembly Method and Photo-Catalytic Property,”
Sciences in China B, Vol. 48, No. 5, May, 2005, pp.
655-659. doi:10.1360/04yb0009
[10] C. Hyeok, S. Elias and D. D. Dionysios, “Photocatalytic
TiO2 Films and Membranes for the Development of Effi-
cient Wastewater Treatment and Reuse Systems,” esali-
nation, Vol. 202, No. 1, Janaury, 2000, pp. 199-206.