Cerium dioxide is a unique material which is promising for biomedical applications. The properties of
ceria are definitely determined by synthesis procedure and further treatment conditions. The vast majority
of currently existing methods of synthesis lead to formation of CeO2 in the form of nanopowders, but in
some cases (e.g., in biological applications), such powders can not be used because they do not quite satisfy
practical requirements. So, in this work we succeed in synthesis of ceria-stable sols with controlled small
particle size (2-4 nm) using an inexpensive and facile method and investigated the effect of changing the
concentrations and the molar ratio of initial reagents on particle size of CeO2. In this paper we also propose
a method of evaluation of toxicity of ceria sols using bioluminescent microorganism Vibrio fischeri. According
to the results obtained CeO2 sols of different concentrations (6.3*10-4 – 0,02M) are not toxic to Vibrio
fischeri.
When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/3495

Baranchikov, A.E.

Ivanov, V.K.

Ivanova, O.S.

Shcherbakov, A.B.

Shekunova, T.O.

SocioEconomic Challenges Journal (Sumy State University)

PROCEEDINGS OF THE INTERNATIONAL CONFERENCE NANOMATERIALS: APPLICATIONS AND PROPERTIES 
Vol. 1 No 2, 02NNBM15(2pp) (2012) 
 
 
2304-1862/2012/1(2)02NNBM15(2) 02NNBM15-1  2012 Sumy State University 
Synthesis of Stable Aqueous Ceria Sols and Study of Their Toxicity 
 
T.O. Shekunova1,*, O.S. Ivanova2,†, A.E. Baranchikov2,‡, V. K. Ivanov1,2,§, A. B. Shcherbakov3,** 
 
1 Moscow State University, Leninskie gory, 119991 Moscow, Russia 
2 Kurnakov Institute of General and Inorganic Chemistry,Russian Academy of Sciences,  
31, Leninskii pr., 119991 Moscow, Russia 
3 Zabolotny Institute of Microbiology and Virusology, National Academy of Sciences of Ukraine, 
154, ul. Zabolotnogo, 03143 Kiev, Ukraine 
 
(Received 13 June 2012; published online 21 August 2012) 
 
Cerium dioxide is a unique material which is promising for biomedical applications. The properties of 
ceria are definitely determined by synthesis procedure and further treatment conditions. The vast majority 
of currently existing methods of synthesis lead to formation of CeO2 in the form of nanopowders, but in 
some cases (e.g., in biological applications), such powders can not be used because they do not quite satisfy 
practical requirements. So, in this work we succeed in synthesis of ceria-stable sols with controlled small 
particle size (2-4 nm) using an inexpensive and facile method and investigated the effect of changing the 
concentrations and the molar ratio of initial reagents on particle size of CeO2. In this paper we also propose 
a method of evaluation of toxicity of ceria sols using bioluminescent microorganism Vibrio fischeri. Accord-
ing to the results obtained CeO2 sols of different concentrations (6.3*10-4 – 0,02M) are not toxic to Vibrio 
fischeri. 
 
Keywords: Ceria, Colloid solutions, Biological activity, Vibrio fischeri. 
 
 PACS numbers: 82.70.Dd, 87.85.Rs 
 
 
______________ 
* tasiok@mail.ru 
† runetta05@mail.ru 
‡ a.barantchikov@gmail.com 
§ van@igic.ras.ru  
** carotene@mail.ru 
1. INTRODUCTION 
 
Cerium dioxide and ceria-based materials are widely 
used in industrial applications, i.e. in manufacturing of 
fuel cells, gas sensors, UV filters, three way catalysts, 
etc. In the last decade, cerium dioxide attracts the atten-
tion of researchers as a potentional inorganic antioxi-
dant. Numerous biomedical applications of ceria-based 
preparations call for nanoceria in the form suitable for 
dosing, i.e., as aggregatively stable CeO2 sols stabilized 
by biocompatible ligands. According to the data of ISI 
Web of Science, the publications on nanobiotechnology 
double every year. Nevertheless, until now the biological 
activity (including biotoxicity) of nanomaterials was 
studied only in isolated cases [1]. Therefore, in the pre-
sent paper, we report a method of synthesis of stable 
colloid solutions of CeO2 nanoparticles with controlled 
particle size. We have also performed an investigation of 
the toxiticy of ceria sols using Vibrio fischeri luminescent 
microorganism. 
 
2. EXPERIMENTAL 
 
To prepare initial solutions, Ce(NO3)3⋅6H2O (rea-
gent grade), citric acid (reagent grade), and aqueous 
ammonia (pure grade) were used. A CeO2 sol was ob-
tained by dissolving calculated amount of reagents in 
25 mL of distilled water (molar ratios of Ce(NO3)3⋅6H2O 
and citric acid were 1:1, 1:2, 1:4, 2:1). Concentrations of 
the reagents were 0.01, 0.025, 0.05, 0.1, 0.2М. The re-
sulting solution was rapidly poured under stirring into 
100 mL of a 3M ammonia solution and allowed to stand 
for 3 h.  
The size and shape of nanoceria particles were deter-
mined by transmission electron microscopy on a Leo 912 
AB Omega electron microscope (100 kV accelerating volt-
age). The optical absorption spectra were recorded on an 
Ocean Optics QE-65000 spectrophotometer in the wave-
length range of 275-900 nm. The size of CeO2 particles 
was measured by the dynamic light scattering method on 
a Malvern Zetasizer Nano ZS analyzer. Before measure-
ments, the sols were diluted with distilled water. The X-
ray powder diffraction of the solid products of the synthe-
sis was performed on a Rigaku D/MAX 2500 diffractome-
ter (CuKα radiation). The size of the coherent scattering 
domains (CSD) was determined by the Scherrer formula. 
The toxicity of sols was assayed by the luminescent 
bacteria test based on the inhibition of their lumines-
cence with different chemical compounds. Standard ly-
ophilized bacteria Vibrio fischeri were used as a biosen-
sor. The intensity of bacterial luminescence (I) was eval-
uated using a Kikkoman Lumitester luminometer. For 
investigation of toxicity, we took two ceria sols with ini-
tial concentrations of 0.005 and 0.02M and molar ratio of 
cerium to citric acid 1:1, and then made a series of dilu-
tions. Final concentrations were 6.3*10-4;1.3*10-3  
2.5*10-3, 5*10-3, 0.01, 0.02M. 
To the resulting ceria sol a calculated amount of 
NaCl was added, so that it concentration was 2%. Re-
hydration of lyophilized Vibrio fischeri bacteria was 
performed by 2% NaCl solution at 4 C for 30 min fur-
ther at 15 C for 30 min. The control sample was a 2% 
solution of NaCl. The bioluminescence intensity was 
measured immediately after mixing of bacteria with 
CeO2 sols and then every 20 min for 2-2.5 h. The dura-
tion of each measurement was 10 s. 
 
T.O. SHEKUNOVA, O.S. IVANOVA, A.E. BARANCHIKOV, ET AL. PROC. NAP 1, 02NNBM15 (2012) 
 
 
02NNBM15-2 
3. RESULTS AND DISCUSSIONS 
 
Ceria forms immediately after mixing initial solu-
tions of cerium(III) nitrate, citric acid, and ammonia, 
which is reflected by the appearance of an absorption 
band with an edge at ~400 nm in the UV-Vis spectrum 
(see Fig. 1). The absorption intensity increases as the 
reaction proceeds because of the increase in the concen-
tration and size of CeO2 nanoparticles in the sol. Our 
data of UV-Vis spectra indicate that, under the condi-
tions used, the formation of the CeO2 phase was com-
pleted in 1.5–2 h. The band gap (Eg) for the resulting sols 
is 3.4-3.5 eV, which noticeably exceeds the Eg value for 
macrocrystalline CeO2 (~3.2 eV) and is evidence of small 
size of CeO2 particles. Eg and the size of particles were 
reanalyzed again after a month. It should be noted that 
the results changed only slightly, indicating a good sta-
bility of sols. 
 
280 320 360 400 440
nm
А
,
В
, min
 
 
Fig. 1 – UV-visible absorption spectra of colloidal ceria solu-
tion formed by mixing aqueous ammonia solutions with 
Ce(NO3)3 (0.025M) and citric acid (0.025M). 
 
According to the X-ray powder diffraction data, all 
samples synthesized were identified as cerium dioxide 
with a fluorite structure and with the size of the coherent 
scattering domain (CSD), calculated from the broadening 
of the diffraction maximum (111), lying in a range of 2.8–
3.5 nm. The transmission electron microscopy (TEM) data 
are in a good agreement with the X-ray powder diffraction 
data. According to TEM (see Fig. 2), CeO2 particles are 
2.0–3.6 nm in size. Analysis of TEM data indicates that 
increasing the concentration of initial reagents does not 
lead to significant change in particle size.  
Dynamic light scattering (DLS) data indicate that 
the hydrodynamic diameter of CeO2 particles for dif-
ferent samples varies from 3 to 6.7 nm. It should be 
noted that the relatively small values of the hydrody-
namic diameters generally indicate a weak aggregation 
of ceria nanoparticles. In addition, according to the 
DLS it can be seen that upon increasing the concentra-
tions of cerium nitrate (III) and citric acid the particle 
size decreases from 6.7 to 3.3 nm. Similarly, upon in-
creasing the molar excess of citric acid from 1:1 to 1:2 
and 1:4, the particle size also decreases from 4.5 to 3 nm. 
 
 
 
Fig. 2 – TEM images of CeO2 nanoparticles in citrate sol. 
 
It was found that CeO2 sol, obtained from mixed 
Ce(NO3)3 (0.05M) and citric acid (0.1M) solution is 
characterized by the smallest particle size and the nar-
rowest particle size distribution. 
Toxicity tests reveal that the luminescence intensity 
of the biosensor placed for 20 min in the NaCl solution 
(2%) is not more than 60-70% of the initial value due to 
inhibition of enzymatic activity of bacteria. According 
to the results obtained on ceria sols, CeO2 nanoparticles 
do not induce any further decrease in the enzymatic 
activity of  Vibrio fischeri, as opposed to ammonium 
citrate stabilizer solution (EC50  0.2M). 
 
4. CONCLUSIONS 
 
In this paper, we have proposed a method of syn-
thesis of cerium dioxide sols stabilized by ammonium 
citrate with controlled particle size. Detailed physico-
chemical attestation of the sols was performed. It was 
established that ceria sols have no toxic effect on Vibrio 
fischeri bacteria.  
 
ACKNOWLEDGMENTS 
 
We are grateful to T.V. Filippova (Moscow State 
University) for the X-ray diffraction analysis of CeO2 
samples.
 
REFERENCES 
 
1. V.K. Ivanov, G.N. Fedotov, M.V. Nikulina, O.S. Polezhaeva, 
G.G. Omel’yanyuk, S.N. Romanenko, S.G. Korol, 
Yu.D. Tret’yakov, Doklady Akademii Nauk 420 No5, 628 
(2008). 


Synthesis of Stable Aqueous Ceria Sols and Study of Their Toxicity

Electronic Sumy State University Institutional Repository

PROCEEDINGS OF THE INTERNATIONAL CONFERENCE NANOMATERIALS: APPLICATIONS AND PROPERTIES Vol. 1 No 2, 02NNBM15(2pp) (2012)   2304-1862/2012/1(2)02NNBM15(2) 02NNBM15-1  2012 Sumy State University Synthesis of Stable Aqueous Ceria Sols and Study of Their Toxicity  T.O. Shekunova1,*, O.S. Ivanova2,†, A.E. Baranchikov2,‡, V. K. Ivanov1,2,§, A. B. Shcherbakov3,**  1 Moscow State University, Leninskie gory, 119991 Moscow, Russia 2 Kurnakov Institute of General and Inorganic Chemistry,Russian Academy of Sciences,  31, Leninskii pr., 119991 Moscow, Russia 3 Zabolotny Institute of Microbiology and Virusology, National Academy of Sciences of Ukraine, 154, ul. Zabolotnogo, 03143 Kiev, Ukraine  (Received 13 June 2012; published online 21 August 2012)  Cerium dioxide is a unique material which is promising for biomedical applications. The properties of ceria are definitely determined by synthesis procedure and further treatment conditions. The vast majority of currently existing methods of synthesis lead to formation of CeO2 in the form of nanopowders, but in some cases (e.g., in biological applications), such powders can not be used because they do not quite satisfy practical requirements. So, in this work we succeed in synthesis of ceria-stable sols with controlled small particle size (2-4 nm) using an inexpensive and facile method and investigated the effect of changing the concentrations and the molar ratio of initial reagents on particle size of CeO2. In this paper we also propose a method of evaluation of toxicity of ceria sols using bioluminescent microorganism Vibrio fischeri. Accord-ing to the results obtained CeO2 sols of different concentrations (6.3*10-4 – 0,02M) are not toxic to Vibrio fischeri.  Keywords: Ceria, Colloid solutions, Biological activity, Vibrio fischeri.   PACS numbers: 82.70.Dd, 87.85.Rs   ______________ * tasiok@mail.ru † runetta05@mail.ru ‡ a.barantchikov@gmail.com § van@igic.ras.ru  ** carotene@mail.ru 1. INTRODUCTION  Cerium dioxide and ceria-based materials are widely used in industrial applications, i.e. in manufacturing of fuel cells, gas sensors, UV filters, three way catalysts, etc. In the last decade, cerium dioxide attracts the atten-tion of researchers as a potentional inorganic antioxi-dant. Numerous biomedical applications of ceria-based preparations call for nanoceria in the form suitable for dosing, i.e., as aggregatively stable CeO2 sols stabilized by biocompatible ligands. According to the data of ISI Web of Science, the publications on nanobiotechnology double every year. Nevertheless, until now the biological activity (including biotoxicity) of nanomaterials was studied only in isolated cases [1]. Therefore, in the pre-sent paper, we report a method of synthesis of stable colloid solutions of CeO2 nanoparticles with controlled particle size. We have also performed an investigation of the toxiticy of ceria sols using Vibrio fischeri luminescent microorganism.  2. EXPERIMENTAL  To prepare initial solutions, Ce(NO3)3⋅6H2O (rea-gent grade), citric acid (reagent grade), and aqueous ammonia (pure grade) were used. A CeO2 sol was ob-tained by dissolving calculated amount of reagents in 25 mL of distilled water (molar ratios of Ce(NO3)3⋅6H2O and citric acid were 1:1, 1:2, 1:4, 2:1). Concentrations of the reagents were 0.01, 0.025, 0.05, 0.1, 0.2М. The re-sulting solution was rapidly poured under stirring into 100 mL of a 3M ammonia solution and allowed to stand for 3 h.  The size and shape of nanoceria particles were deter-mined by transmission electron microscopy on a Leo 912 AB Omega electron microscope (100 kV accelerating volt-age). The optical absorption spectra were recorded on an Ocean Optics QE-65000 spectrophotometer in the wave-length range of 275-900 nm. The size of CeO2 particles was measured by the dynamic light scattering method on a Malvern Zetasizer Nano ZS analyzer. Before measure-ments, the sols were diluted with distilled water. The X-ray powder diffraction of the solid products of the synthe-sis was performed on a Rigaku D/MAX 2500 diffractome-ter (CuKα radiation). The size of the coherent scattering domains (CSD) was determined by the Scherrer formula. The toxicity of sols was assayed by the luminescent bacteria test based on the inhibition of their lumines-cence with different chemical compounds. Standard ly-ophilized bacteria Vibrio fischeri were used as a biosen-sor. The intensity of bacterial luminescence (I) was eval-uated using a Kikkoman Lumitester luminometer. For investigation of toxicity, we took two ceria sols with ini-tial concentrations of 0.005 and 0.02M and molar ratio of cerium to citric acid 1:1, and then made a series of dilu-tions. Final concentrations were 6.3*10-4;1.3*10-3  2.5*10-3, 5*10-3, 0.01, 0.02M. To the resulting ceria sol a calculated amount of NaCl was added, so that it concentration was 2%. Re-hydration of lyophilized Vibrio fischeri bacteria was performed by 2% NaCl solution at 4 C for 30 min fur-ther at 15 C for 30 min. The control sample was a 2% solution of NaCl. The bioluminescence intensity was measured immediately after mixing of bacteria with CeO2 sols and then every 20 min for 2-2.5 h. The dura-tion of each measurement was 10 s.  T.O. SHEKUNOVA, O.S. IVANOVA, A.E. BARANCHIKOV, ET AL. PROC. NAP 1, 02NNBM15 (2012)   02NNBM15-2 3. RESULTS AND DISCUSSIONS  Ceria forms immediately after mixing initial solu-tions of cerium(III) nitrate, citric acid, and ammonia, which is reflected by the appearance of an absorption band with an edge at ~400 nm in the UV-Vis spectrum (see Fig. 1). The absorption intensity increases as the reaction proceeds because of the increase in the concen-tration and size of CeO2 nanoparticles in the sol. Our data of UV-Vis spectra indicate that, under the condi-tions used, the formation of the CeO2 phase was com-pleted in 1.5–2 h. The band gap (Eg) for the resulting sols is 3.4-3.5 eV, which noticeably exceeds the Eg value for macrocrystalline CeO2 (~3.2 eV) and is evidence of small size of CeO2 particles. Eg and the size of particles were reanalyzed again after a month. It should be noted that the results changed only slightly, indicating a good sta-bility of sols.  280 320 360 400 440nmА,В, min  Fig. 1 – UV-visible absorption spectra of colloidal ceria solu-tion formed by mixing aqueous ammonia solutions with Ce(NO3)3 (0.025M) and citric acid (0.025M).  According to the X-ray powder diffraction data, all samples synthesized were identified as cerium dioxide with a fluorite structure and with the size of the coherent scattering domain (CSD), calculated from the broadening of the diffraction maximum (111), lying in a range of 2.8–3.5 nm. The transmission electron microscopy (TEM) data are in a good agreement with the X-ray powder diffraction data. According to TEM (see Fig. 2), CeO2 particles are 2.0–3.6 nm in size. Analysis of TEM data indicates that increasing the concentration of initial reagents does not lead to significant change in particle size.  Dynamic light scattering (DLS) data indicate that the hydrodynamic diameter of CeO2 particles for dif-ferent samples varies from 3 to 6.7 nm. It should be noted that the relatively small values of the hydrody-namic diameters generally indicate a weak aggregation of ceria nanoparticles. In addition, according to the DLS it can be seen that upon increasing the concentra-tions of cerium nitrate (III) and citric acid the particle size decreases from 6.7 to 3.3 nm. Similarly, upon in-creasing the molar excess of citric acid from 1:1 to 1:2 and 1:4, the particle size also decreases from 4.5 to 3 nm.    Fig. 2 – TEM images of CeO2 nanoparticles in citrate sol.  It was found that CeO2 sol, obtained from mixed Ce(NO3)3 (0.05M) and citric acid (0.1M) solution is characterized by the smallest particle size and the nar-rowest particle size distribution. Toxicity tests reveal that the luminescence intensity of the biosensor placed for 20 min in the NaCl solution (2%) is not more than 60-70% of the initial value due to inhibition of enzymatic activity of bacteria. According to the results obtained on ceria sols, CeO2 nanoparticles do not induce any further decrease in the enzymatic activity of  Vibrio fischeri, as opposed to ammonium citrate stabilizer solution (EC50  0.2M).  4. CONCLUSIONS  In this paper, we have proposed a method of syn-thesis of cerium dioxide sols stabilized by ammonium citrate with controlled particle size. Detailed physico-chemical attestation of the sols was performed. It was established that ceria sols have no toxic effect on Vibrio fischeri bacteria.   ACKNOWLEDGMENTS  We are grateful to T.V. Filippova (Moscow State University) for the X-ray diffraction analysis of CeO2 samples. REFERENCES  1. V.K. Ivanov, G.N. Fedotov, M.V. Nikulina, O.S. Polezhaeva, G.G. Omel’yanyuk, S.N. Romanenko, S.G. Korol, Yu.D. Tret’yakov, Doklady Akademii Nauk 420 No5, 628 (2008). 

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Synthesis of Stable Aqueous Ceria Sols and Study of Their Toxicity

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