J Sol-Gel Sci Technol (2008) 48:66–72
DOI 10.1007/s10971-008-1753-9
ORIGINAL PAPER
Utilization of sol–gel ceramics for the immobilization of living
microorganisms
U. Soltmann Æ H. Böttcher
Received: 15 January 2008 / Accepted: 18 April 2008 / Published online: 12 May 2008
Ó Springer Science+Business Media, LLC 2008
Abstract Two possible methods are described for using
sol–gel technology to immobilize living microorganisms,
either embedding the cells within thin silica layers, or using
the technique of freeze-gelation to immobilize microor-
ganisms within molded ceramic parts. The preparation and
structure of both biocer variants are outlined, and examples
are given for the activity and storage stability of embedded
microorganisms. Silica layers were used to immobilize
various bacteria and algae. Survival rates after storage
are given for Pseudomonas fluorescence bacteria and for
green algae such as H. pluvialis, C. vulgaris, B. braunii
and N. limnetica embedded within thin transparent silica
layers. The bioactivity of bacteria immobilized in freeze-
gelation ceramics was investigated by monitoring glucose
consumption for P. fluorescens NCIMB 11764, and phenol
degradation for Rhodococcus ruber.
Keywords Biocers
Sol–gel
Silica coatings

Freeze-gelation
Immobilization
Algae
Bacteria

Spores
1 Introduction
For various biotechnology applications, it is advantageous
to immobilize living cells within a stable matrix system,
thus making efficient use of their physiological capabilities,
e.g., for producing secondary metabolites, or in biotrans-
formation/biocatalysis reactions. The primary benefits of
U. Soltmann (&)
H. Böttcher
Gesellschaft zur Förderung von Medizin-, Bio- und
Umwelttechnologien e.V., GMBU, P.O. Box 520165, 01317
Dresden, Germany
e-mail: soltmann@gmbu.de
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immobilization are the prevention of culture-washout,
easier separation of formed products, and the protection of
embedded cells from e.g., shear forces, pH, or solvents.
However, immobilization often limits mass transfer and
also can hinder cell division inside the immobilization
matrix. Besides commonly used natural polymers like, e.g.,
alginate sol–gel technology has become increasingly
important for immobilizing living cells such as bacteria,
yeasts, and plant or animal cells [cf. 1–8].
Combining a ceramic-like oxide matrix with biological
systems (‘‘biocers’’ [1]) offers important advantages. Such
biocomposites hold promise for technical applications due
to their combination of the mechanical, chemical, thermal,
and photochemical stability of the inorganic host matrix
with the high variability of the sol–gel process (e.g.,
chemical modifications, tailored porosity) and the broad
range of applicative forms of sol–gel derived materials
(e.g., coatings, granules, shaped bulk products).
Conventional sol–gel procedures generally involve
extreme pH-values and high concentrations of alcohol,
each usually fatal to living cells. Therefore, the sol–gel
transition must be carried out under physiological condi-
tions using mainly neutral aqueous nanosols followed by
mild solidification and drying processes.
The resulting viability is affected to a large degree by
the shrinkage, pore structure, and rigidity of the matrix.
These factors can be controlled by the drying regime (air-
or freeze-drying), the residual water content, the addition
of fillers (e.g., ceramic fibers) [9], the admixture of soluble
and leachable pore-forming additives (e.g., sorbitol) [10],
or the incorporation of water-retaining polymers such as
polyethylene glycol [11].
However the metabolic activity of the embedded cells
and their storage stability depends mainly of the type and
physiological state of the embedded cells. Normally the
J Sol-Gel Sci Technol (2008) 48:66–72
metabolic activity of non-growing cells decreases over the
time. Thus to achieve constant activity over a lengthy time,
the deactivation of cells must be compensated for by cell
growth. This balance can be promoted by a periodic change
between growth and non-growth conditions. Therefore the
immobilization matrix should permit cell division in order
to compensate for the loss of activity of embedded cells.
In this article two variants of biocer preparation will be
described. First the immobilization of living cells within
thin transparent silica layers. This is a simple means of
immobilizing cells on various material surfaces. Depend-
ing on the type of the cells and their dimensions, and on
the thickness of the silica layer, immobilized cells are able
to grow out of the rigid silica layer. The second variant is
to embed microorganisms within molded ceramic parts
using the freeze-gelation technique, whereby microor-
ganisms are immobilized in a conserved state within
porous, shaped ceramic composites. This latter preparation
method results in a directional porosity, with pores formed
in the lm range allowing for cell division inside of the
biocer.
2 Experimental
2.1 Biocomponents
Pseudomonas fluorescens strain NCIMB 11764 was culti-
vated at 30 °C in minimal glucose–ammonia medium
consisting of 0.05 g/L NH4Cl, 8.71 g/L KH2PO4, 0.40 g/L
MgSO4
7H2O, 0.01 g/L FeSO4
7H2O, and 3.60 g/L
glucose. Rhodococcus ruber cells, an isolate from the mud
of the Saale river (Germany), were grown at 30 °C in
mineral medium (DSMZ media number 457 with trace
element solutions SL-4 and SL-6) containing 500 mg/L
phenol. The bacteria were cultivated to the end of their
exponential growth phase before using them for immobi-
lization experiments. Bacterial cell numbers were
determined by the number of colony forming units (CFU)
after plating of gradually diluted bacteria suspensions on
nutrient agar.
Haematococcus pluvialis strain 192.80 and Nanno-
chloropsis limnetica strain 18.99 were obtained from SAG
(Culture Collection of Algae/University of Göttingen) and
cultivated using a semi-synthetic full medium [12] or the
medium EsAg (SAG) in case of N. limnetica. Botryococcus
braunii 807/1 obtained from CCAP (Culture Collection
of Algae and Protozoa, Scotland, UK) were cultivated
on Bold-Basal Medium 3N-BBM + V (CCAP); and for
Chlorella vulgaris algae Tamiya medium [13] were used.
The algae were cultivated at room temperature and illu-
minated at 120 lE * m-2s-1 with a light to dark
photoperiod of 16–8 h.
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2.2 Preparation of the silica sols
Sol A: 10 mL TEOS (tetraethoxysilane, Fluka), 60 mL
H2O, 30 mL 0.01 M HCl are stirred at ambient temperature
for 20 h resulting in an acidic nanosol with approximately
3 wt% SiO2 in 10% ethanol.
Sol B: 9 mL TEOS, 1 mL GLYEO [(3-glycidyloxy-
propyl)-triethoxysilane, Fluka], 60 mL H2O, 30 mL
0.01 M HCl are stirred for 20 h at ambient temperature
resulting in an acidic nanosol with approximately 3.5 wt%
solid content in 10% ethanol.
2.3 Preparation of the biocers
Thin silica layers with embedded microorganisms were
prepared by dip-coating of glass slides at a constant pulling
rate of 300 mm/min in sol A or B containing the micro-
organisms. Before adding the cells to the silica sols the pH
was increased up to pH 7 by adding of 1 M NaOH. After
coating the glass slides were air-dried for about 15 min and
subsequently transferred into nutrient solution or stored in
a wet atmosphere at 4 °C.
Freeze-gelation biocers were prepared by mixing of
90 g mullite (Mullite73, Osthoff-Petrasch, Hamburg) and
45 g KöstrosolÒ 3550 (Chemiewerke Bad Köstritz) to a
slurry. Pseudomonas fluorescence and R. ruber bacteria
were cultivated to the end of their exponential growing
phase and harvested by centrifugation at 3,000 rpm. Two
milliliters of concentrated bacteria solution, equivalent to
82 mg P. fluorescence bacteria (dry weight) or 134 mg in
case of R. ruber, were admixed to the slurry. The slurries
were poured into moulds and frozen at -80 °C. Finally,
after demolding the biocers were dried by lyophilization.
2.4 Characterization of the biocers
Light and fluorescence microscopy were used to estimate the
vitality of immobilized microorganisms in silica layers.
Embedded bacteria were characterized by the intactness of
the cell membranes using the LIVE/DEAD BacLight via-
bility kit (Invitrogen-Molecular Probes). The vitality of
immobilized algae was determined by their pigmentation,
with pigmented cells being alive and unpigmented ones dead.
Scanning electron micrographs were taken at 1–5 kV using
a Gemini 982 scanning electron microscope (LEO, Oberko-
chen). The samples were embedded in liquid colloidal silver
on conductive carbon sheets and shadow casting was made
with carbon (Baltec MED 010, BAL-TEC, Liechtenstein).
Glucose concentrations were measured colorimetrically
using an enzymatic test kit (Glucose liquicolor, HUMAN,
Wiesbaden, Germany). The phenol concentrations were
determined by the dye formation with 4-aminoantipyrene
and potassium hexacyanoferrate [14].
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3 Results and discussion
3.1 Thin silica layers with embedded microorganisms
3.1.1 Preparation of thin biocer layers
Embedding biocomponents within thin silica layers is a
cost-efficient way of producing bioactive materials. The
coating of large surfaces requires relatively small amounts
of sol and biocomponents. Figure 1 illustrates how bio-
components can be embedded within a sol–gel layer.
For a mild preparation of thin layers with embedded
living cells, neutral aqueous nanosols are necessary. The
alcohol formed by alkoxide hydrolysis can be evaporated
by passing air through the solution and simultaneously
substituted for by water, as described for the sol–gel
immobilization of bone-relevant scleroproteins in 2000
[15]. Sols (A + B) had a low solids content with small,
mostly non-aggregated particles of mean size 5–10 nm that
showed excellent adhesion and film-formation properties
on different substrates. After the nanosol was neutralized,
the biocomponents were admixed into it. Thin sol–gel films
can be produced without any problems using conventional
deposition technologies such as dip, spin or spray coating.
Strongly adhering xerogel films are formed after drying.
The degree to which the embedded cells are immobilized is
determined by the thickness of the sol–gel film, the type
and dimensions of the cells, the biomass:oxide ratio, and
the admixture of additives such as nutrients, humidity
preserver, or penetration agents.
Figure 2 shows Pseudomonas fluorescence and Rhodo-
coccus rhodochrous bacteria, and Chlorella vulgaris algae
embedded within thin silica layers prepared using Sol A.
Since layer thickness is approximately 0.3–1.0 lm,
immobilized cells are easily accessible to external reagents.
3.1.2 Silica layers with embedded bacteria
Coated glass substrates with immobilized Rhodococcus
rhodochrous, Aspergillus versicolor, or Bacillus sphaericus
cells were successfully tested for their ability to biodegrade
Fig. 1 Preparation of silica
layers with embedded
microorganisms
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J Sol-Gel Sci Technol (2008) 48:66–72
either phenols or glycols, or used in biosorption experi-
ments, as previously described [9, 16, 17].
The applicability of biocer layers is limited by the poor
storage capability of immobilized vegetative cells, as drying
out of the embedded cells affects the integrity of the mem-
brane. For example, 30% of embedded R. rhodochrous cells
and 70% of embedded B. sphaericus cells died within 3 days
when stored at 4 °C in a wet atmosphere [9, 16].
Currently bioremediation experiments were performed
with P. fluorescence in the lab with waters containing
cyanide. Pseudomonas fluorescence is able to use cyanide
as a source of nitrogen. First experiments to the survival of
embedded cells (live/dead staining) showed that after 48 h
storage at 4 °C in a humid atmosphere outside of liquid
culture media approximately 35% of the embedded cells
were still alive.
A way of circumventing the poor storage stability of
microorganisms is to use spores. These very robust resting
cells can survive harsh preparation conditions yet still show
high germination rates even after long-term storage at room
temperature and under dry conditions. For example, Asper-
gillus versicolor or Bacillus sphaericus spores embedded
within thin silica layers still maintained their ability to ger-
minate after being stored for 2.5 years at room temperature
under dry conditions [9, 16]. On being transferred into
nutrient solution, they grew out from the layer to form a
biofilm on the surface of the coated material. In the case of
B. sphaericus spores (Fig. 3), encapsulation within the silica
matrix did inhibit nutrient-induced germination, but using a
chemical trigger system of Ca2+ and DPA made possible a
selective induction of the germination of the embedded
spores [16].
3.1.3 Silica layers with embedded microalgae
An interesting field of application is the use of sol–gel
layers to immobilize microalgae. Due to the transparency
of the silica layer, the immobilization matrix does not
affect the photosynthetic activity of the algae. Immobilized
algae could be advantageous for optimally designing pho-
tobioreactors and for biosensors. By using immobilized
J Sol-Gel Sci Technol (2008) 48:66–72 69

Fig. 2 SEM micrographs of

Pseudomonas fluorescence (a)
and Rhodococcus rhodochrous
(b) bacteria and a 3D digital
microscopic image of a
Chlorella vulgaris algae (c)
embedded within thin silica
layers

Fig. 3 Scanning electron

microscopic images of coated
glass substrates with embedded
B. sphaericus JG-A12 spores:
(a) before germination and (b)
outgrowing vegetative cell

algae cells within photobioreactors, a high light efficiency

could be realized and thus process control simplified, e.g.,
applying stress factors to induce the production of sec-
ondary metabolites or the removal of formed products.
Recently the use of sol–gel immobilized Haematococ-
cus pluvialis algae for the production of astaxanthin was
reported [12]. Up to 105 cells/cm2 could be immobilized,
while still maintaining a layer with sufficient mechanical
stability. The immobilized cells remained viable for more
than 40 days. It was shown that the astaxanthin formed
could be extracted from the immobilized cells using vari-
ous solvents. By co-immobilizing glycerol within the
layers as a cell-protective additive, about 30% of the
embedded algae cells survived the short contact time with
glycol ethers, the solvent required to extract astaxanthin. Fig. 4 Percentage of viable immobilized algae cells on coated glass
At present various algae are being tested regarding their samples after storage at 4 °C in the dark
behavior (long-term activity, cell division, and storage sta-
bility) within thin silica layers. Figure 4 shows initial results
for the storage stability of immobilized Chlorella vulgaris, 3.2 Freeze-gelation biocers
Haematococcus pluvialis, Botryococcus braunii, and Nan-
nochloropsis limnetica algae. The algae were coated on 3.2.1 Preparation and structure of freeze-gelation biocers
glass slides using sol B then stored separately in the dark at
4 °C in 50 mL test tubes with culture medium. Periodically The freeze-gelation (or freeze-casting) technique allows
samples were taken and characterized by light microscopy. the cost-effective production of near net-like ceramics
After 25 days storage, or in the case of Botryococcus where porosity can be tailored for applications such as
12 days, 80–100% of the embedded algae remained viable. filters, catalyst supports, and substrates for biotechnical use
Thus easy handling of the immobilized algae is possible. [18–20]. Since the preparation procedure uses aqueous
Investigations are ongoing into these new opportunities to ceramic powder slurries and since the non-sintered com-
use immobilized algae for the efficient photosynthetic pro- posite has adequate mechanical strength, the method is
duction of valuable metabolic products such as unsaturated favored for embedding living microorganisms within
fatty acids and long-chain hydrocarbons. ceramic matrices [21].

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Fig. 5 Preparation of freeze-
gelation biocers
As shown in Fig. 5, freeze-gelation biocers are pre-
pared by making a slurry from aqueous nanosols, ceramic
powder, and the biocomponent together with additives
such as nutrients and cryoprotectives. This is poured into
a mold and subsequent solidification is attained by
freezing. The formation of ice crystals causes phase
separation and the forced packing of the nanoparticles
results in the sol–gel transition. Finally, after demolding,
drying takes place by lyophilization or evaporation. By
combining freezing and lyophilization, the embedded
microorganisms are conserved in a freeze-dried state.
Consequently, they can be stored for long periods of time
under dry conditions without a substantial loss of viability
[21].
The microstructure and porosity of freeze-gelation
ceramics are determined by how ice-crystals form during
freezing. This can be affected by the rate of cooling, the
water content of the slurry, the mass fraction and size ranges
of the filler particles, and by the choice of nanosol. Addi-
tionally, the resulting structure is influenced by the
admixture of the biocomponent and additives. It is necessary
to optimize the freezing conditions to achieve a suitable
combination of cell survival rate and desired porosity for a
given biocomponent. Figure 6 shows some images of the
structural conditions of freeze-gelation ceramics.
SEM image b shows the cross-section of the wall of a
hollow cylinder. A directed porosity can be seen, with very
small pores at the surface and pores in the lm range inside
of the matrix as a result of the freezing conditions. The
freezing front starts at the cooled surface and, due to the
high cooling rate, very small ice crystals form near the
surface. Inside the biocer where the cooling rate is lower,
larger ice crystals are formed that result in larger pores.
More details of the porous structure are shown in images
c–f. In image f the nanoparticulate structure of the com-
posite can be seen, with the aggregated single nanoparticles
of the sol and the mesoporosity of the material. When
freeze-gelation is used, embedded cells can be easily
accessed by external reagents and, due to the large pores
inside, cell division of the embedded cells is possible.
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J Sol-Gel Sci Technol (2008) 48:66–72
3.2.2 Freeze-gelation ceramics with embedded R. ruber
and P. fluorescence cells
Rhodococcus ruber and Pseudomonas fluorescence bacte-
ria were used for immobilization experiments. The strain
R. ruber is capable of degrading phenols very efficiently
while the strain P. fluorescence can degrade cyanide ions
in wastewater. Freezing, a key step in generating freeze-
gelation ceramics has a strong impact on the survival rate
of embedded cells. Depending on the rate of cooling, via-
bility is affected by the formation of ice crystals inside of
the cells or by high intercellular salt concentrations
resulting from the efflux of water. For example, only 9% of
Rhodococcus ruber and 0.3% of Pseudomonas fluores-
cence bacteria survive the freezing and drying conditions
used in preparing freeze-gelation biocers.
The use of such cryoprotective agents as sugars or
glycerol allows higher survival rates to be realized [22].
The first papers describing the effects of glycerol and
DMSO were published as early as the 1940s and 50s [23,
24]. Recently, Tessema et al. [25] described the protective
effect of trehalose and glycerol on freeze-dried, sol–gel
immobilized, genetically engineered E. coli cells. Table 1
shows the effect of different sugars, at concentrations up to
30%, on the survival rate of P. fluorescence and R. ruber.
With the addition of trehalose, a sevenfold higher survival
rate of R. ruber could be attained. For P. fluorescence best
results were obtained when 15% lactose was added, resulting
in a 58-fold higher survival rate. However such high sugar
concentrations are problematic when making freeze-gela-
tion biocers. They influence the formation of ice-crystals,
hinder the formation of the silica gel network, and destabi-
lize the matrix by successive leaching of the sugar. When
immobilizing very sensitive bacteria, pre-immobilization of
the cells and cryoprotectives into very small alginate beads
then subsequently embedding such beads into a freeze-
gelation matrix could be a means of avoiding such problems.
Estimating the number of surviving bacteria within a
freeze-gelation matrix was problematic. Direct microscopic
studies were not possible as the matrix was not transparent,
J Sol-Gel Sci Technol (2008) 48:66–72 71

Fig. 6 Photograph of freeze-

gelation ceramics and SEM
images of the cross-section of
the wall of a hollow cylinder at
increasing magnification

Table 1 Effect of different sugars on the survival rate of non-immobilized R. ruber and P. fluorescence cells after freeze-drying
Cryo-protective R. rubber P. fluorescence
agent
Sugar Survival Sugar Survival
concentration (%) rate (%) concentration (%) rate (%)

Without 0 0.9 0 0.03

Lactose 20 1.7 15 1.9
Fructose 20 1.0 10 1.6
Trehalose 20 6.1 10 0.6
Sorbitol 20 1.0 10 1.0
Mannitol 10 1.7
Saccharose 20 1.0

while cultivation-based cell counting techniques (colony

forming units), which require a disintegration of the matrix,
underestimated the number of cells. Therefore the meta-
bolic activity of embedded P. fluorescence or R. ruber
bacteria was tested for freeze-gelation biocers made with-
out the addition of cryprotectives. The experiments were
performed under batch conditions. The nutrient solution
was renewed regularly if the nutrients became exhausted or
if the growth of non-immobilized cells became visible.
Figure 7 shows glucose consumption by freeze-gelation
biocers with embedded P. fluorescence bacteria. There was
a strong increase in glucose consumption over time. During
the experiment the surface of the biocer became covered
with pseudomonas cells. After 35 days the surface of the
biocer body was cleaned. Although the activity decreased,
because of the cells within the biocer it was still higher than
initially. The porous structure permits good transport of
Fig. 7 Glucose consumption by embedded P. fluorescence cells
oxygen and nutrients, and it seems that cell division occur-
ring within the matrix compensated for the freeze-induced
loss of viability. The structure of freeze-gelation biocers rapidly when the freeze-dried biocers were transferred into
remained unaffected over the testing time of 45 days. a phenol-containing nutrient solution and there was a clear
Table 2 shows the rate of phenol degradation by increase in the rate of phenol degradation in the first few
embedded R. ruber bacteria. The embedded cells responded days.

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Table 2 Change in the rate of phenol degradation by R. ruber
embedded within a freeze-gelation matrix
Operation Phenol degradation
time (days) rate (mg/d 9 g biocer)
0 0.61
6 1.27
13 1.05
21 1.51
28 1.50
The freeze-dried R. ruber biocers can be stored under
dry conditions at least for a time of 21 days without loss of
activity or response.
4 Conclusions
Silica layers with immobilized bacteria, fungi, or algae can
easily be prepared using conventional deposition tech-
niques such as dip-coating. Since the silica layer thickness
is between 0.3 and 1 lm, cells are either completely or
partially covered by the silica layer, depending on the
dimension of the immobilized microorganisms. Due to the
transparency of the silica matrix, photosynthetic microor-
ganisms such as algae can be immobilized to advantage.
The cells are easy accessible to external reagents, but when
stored out of liquid media at low temperatures and in a
humid atmosphere, the embedded cells died within a few
days. Storage-stable layers can be fabricated using spores.
Embedded A. versicolor or B. sphaericus spores still retain
their ability to germinate after storage for 2.5 years under
dry conditions.
The freeze-gelation technique can be used to immobilize
and conserve living cells within shaped bulk products. The
conditions of freezing determine the microstructure and
porosity of the composite, and have a strong impact on the
survival rate of embedded microorganisms. In the case of
R. ruber and P. fluorescens cells, only 9% and 0.3%,
respectively, of the embedded cells survive the freezing
and freeze-drying steps. The directional growth of ice
crystals results in the formation of pores in the lm range
within the biocers. This makes the embedded biocompo-
nents easily accessible, and enables cell division to take
place within the biocers. Those cells surviving in the dry
biocers showed a fast response and increase in metabolic
activity on being transferred into nutrient solution.
By the combination of sol–gel matrices with living
microorganisms an optimized use of the physiological
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J Sol-Gel Sci Technol (2008) 48:66–72
capabilities of the cells for applications in bioremediation,
the production of valuable products or biosensors seems
possible. Nevertheless, further research to improve the
lifetime, response characteristic and to control a defined
cell status within the matrices is necessary.
Acknowledgments The authors would like to thank Dr. S. Matys
and Dr. M. Mkandawire, Institute for Material Science, Dresden
University of Technology, for the SEM micrographs. The work was
supported by the German Ministry of Education and Research (project
03WKBH6B & 02WR0696).
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