Biomimetic Control of Size in the Polyamine-Directed Formation of Silica Nanospheres

Article abstract of DOI:10.1002/anie.200352212

Diatom shells (see picture of cell of Stephanopyxis turris) contain long-chain polyamines attached either to putrescine or to phosphorylated silaffin peptides. These polyamines direct the in vitro formation of silica nanospheres from silicic acid. The con

Full text of DOI:10.1002/anie.200352212

Communications
Biomineralization
Biomimetic Control of Size in the Polyamine-
Directed Formation of Silica Nanospheres**
Manfred Sumper,* Sonja Lorenz, and Eike Brunner
The intricate silicified cell walls of diatoms are probably the
most outstanding examples of nanoscale-structured materials
[
1]
in nature. The degree of complexity in these hierarchically
organized biominerals has never been matched in artificial
[
2]
materials. Nanofabrication of silica in diatoms occurs under
ambient conditions at slightly acidic pHvalues and results
from specific interactions between biomolecules and silicic
acid derivatives. Such biomolecules are the silaffin peptides
and long-chain polyamines (both of which were isolated from
[
3–5]
diatom biosilica
) and the silicateins (isolated from
[
6]
sponges). Silaffins isolated from Cylindrotheca fusiformis
mainly consist of modified lysine and serine residues. The
serine groups are phosphorylated and the lysine residues are
[*] Prof. Dr. M. Sumper
Lehrstuhl Biochemie I, Universität Regensburg
93053 Regensburg (Germany)
Fax: (+49)941-943-2936
E-mail: manfred.sumper@vkl.uni-regensburg.de
S. Lorenz, Prof. Dr. E. Brunner
Institut für Biophysik und physikalische Biochemie
Universität Regensburg
93053 Regensburg (Germany)
[
**] We thank R. Deutzmann and E. Hochmuth for MS analyses and R.
Fischer for technical assistance. The present work was supported by
the Deutsche Forschungsgemeinschaft (grants SFB 521A2 and -A6)
and the Fonds der Chemischen Industrie.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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DOI: 10.1002/anie.200352212
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Chemie
converted into three different derivatives: e-N-dimethyl-
lysine, phosphorylated e-N-trimethyl-d-hydroxylysine, and
lysines covalently linked to long-chain polyamines, N-methyl-
lag phase, silica started to precipitate (Figure 1a, blue line).
However, this precipitation was strictly dependent on the
presence of phosphate. In the presence of acetate anions only
(the buffer system), no precipitate was formed at all
(Figure 1a, green line). Silica precipitation in the presence
of increasing phosphate concentrations produced nano-
spheres with increasing diameters (Figure 1b, red line). It is
the particle size of the silica nanospheres that is strictly
controlled by the concentration of phosphate anions. Defined
particle diameters between 30 and 700 nm were obtained and
the resulting size distributions were close to monodisperse.
Replacement of orthophosphate by pyrophosphate, an anion
[
7,8]
ated derivatives of a polypropyleneimine.
Silaffins as well
as long-chain polyamines have been shown to rapidly direct
[
3,4]
the formation of silica nanospheres from silicic acid in vitro
[
9]
which has been confirmed and adapted to technical
[
10]
applications.
In the past, a huge amount of knowledge has been
accumulated with respect to the inorganic chemistry of silicic
[
11]
acid and its polymeric derivatives. Nonporous silica nano-
spheres could be synthesized by controlled hydrolysis of
[
12]
silicon alkoxides in methanol/NH mixtures and the under-
3
[
13,14]
lying mechanism of size control has been investigated.
The past decade has seen important advances in the ability to
fabricate porous solids and several methods have been
developed for the synthesis of different silica morphologies
[
2,15,16]
summarized in recent reviews.
By applying this knowl-
edge to the unique structure of silaffins, a fascinating aspect
emerges: Nature has designed a molecule that is perfectly
adapted to the chemistry of silica formation, as briefly
outlined. Silicic acid polymerization involves three distinct
stages. First, monomeric silicic acid polymerizes by conden-
sation of silanol groups to form dimers, trimers, and cyclic
oligomers. Oligosilicic acid species have a strong tendency to
polymerize further in such a way that siloxane-bond (SiÀOÀ
Si) formation is maximized. These early processes create
highly branched polysilicic acids as nuclei for silica formation.
Second, the nuclei grow to form spherical particles either by
continuous polymerization with monomeric and oligomeric
silicic acids or by fusion of particles. Finally, the silica
nanospheres can precipitate by flocculation, a process that
intimately connects silica spheres and produces hard silica, as
is found in the cell wall of diatoms. The structural elements
found in silaffins are likely to be involved in each of these
three stages, thereby accelerating the formation of hard silica
by orders of magnitude. More than 40 years ago, quaternary
ammonium ions were recognized as structure-directing agents
[
17]
in the synthesis of zeolite molecular sieves.
methylammonium cation favors the formation of symmetric
The tetra-
8
À
oligosilicate anions such as the cubic octamer Si O , and
8
16
this control of silicate speciation influences the nucleation
[
18]
phase of silica formation.
Owing to their unique e-N-
Figure 1. Characteristics of silica fabrication catalyzed by the polya-
mine/phosphate system. a) Kinetics of silica precipitation. The incuba-
tion mixture contained sodium phosphate (pH 5.5; 30 mm), polyamine
(0.2 mm), and mono-/disilicic acid (40 mm) at 258C. After the times
indicated, aliquots (50 mL) were removed and centrifuged. Precipitated
silica was dissolved in NaOH (2m; 5 min at 908C) and quantified by
trimethyl-d-hydroxylysine residue, silaffins may exert a
similar control. Polyamines were shown to catalyze the
[
19]
polycondensation of silanol groups and are known to act
[
11]
as efficient flocculation agents. Recently, an additional type
of modification, a high degree of phosphorylation of silaffins,
was shown to be essential for the formation of silica nano-
[
11]
the molybdate method. An absorbance of 1.0 corresponded to
.5 mmol SiO . Blue line: All components were mixed at t=0 min.
0
[
7]
2
spheres in vitro.
Green line: Phosphate was replaced by sodium acetate (pH 5.5;
0 mm). Red line: Silicic acid was preincubated for 15 min in sodium
Herein, we define the function of phosphate anions in the
polyamine-directed formation of silica nanospheres with the
polyamines extracted from the cell wall of the diatom
Stephanopyxis turris as a model. These polyamines consist
of 15–21 N-methylpropyleneimine repeating units attached to
putrescine. The polyamines were able to precipitate silica
nanospheres from a silicic acid solution after a few minutes,
even under acidic conditions that presumably represent the
3
phosphate (pH 5.5; 30 mm) and polyamine was added at t=0 min.
b) Silica nanosphere diameters as a function of the multivalent anion
concentration. Silica fabrication was performed in a sodium acetate
buffer (pH 5.5; 30 mm) as in part a) with increasing concentrations of
multivalent anions. The reaction was allowed to proceed for 12 min.
The resulting nanospheres were collected by centrifugation and ana-
lyzed by SEM. Response to orthophosphate and pyrophosphate is
shown by the red and the blue lines, respectively. Insets: SEM micro-
graphs of the corresponding precipitates. Scale bars: 1000 nm.
[
20]
physiological situation (approximately pH5). After a short
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Communications
with a higher negative charge, gave rise to a drastic effect.
Control of nanosphere size distribution was exerted at anion
concentrations nearly two orders of magnitude lower and the
maximum sphere diameter was increased to about 1000 nm
(
Figure 1b, blue line). Importantly, other multivalent anions
can produce silicate precipitates as well, whereas monovalent
anions such as chloride or acetate ions fail to do so. In
particular, we have tested sulfate ions and polyvalent anions
such as DNA and sodium dodecyl sulfate (see Supporting
Information). The shape of the precipitates formed in the
presence of the latter anions, however, is strongly different
from the spherical nanoparticles obtained in the presence of
phosphate ions.
Long-chain polyamines from diatom biosilica behave like
amphiphilic substances. They are extractable from an aqueous
solution by chloroform/methanol (3:2 v/v). Possibly, these
polyamines form aggregates in aqueous solution (micro-
emulsions) with positively charged surfaces. If so, multivalent
anions should promote higher-order assemblies of the emul-
sion droplets. This possibility was investigated by NMR
spectroscopy and dynamic light scattering techniques. The
1
pronounced influence of phosphate ions on the HNMR
spectrum of polyamines from S.turris is demonstrated in
1
Figure 2a. In the presence of phosphate (top trace), the H
nuclei attached to the repeating unit give rise to three
signals A, B, and C. In the absence of phosphate, these signals
split into two or more components (bottom trace). Evidently,
the polyamine molecules exist in different states in the
absence of phosphate, whereas a unique state is observed for
1
the phosphate-containing sample. Furthermore, the HNMR
chemical shifts of the signals, except for C1 and C2, strongly
depend on the type and concentration of the anions added, as
shown for signal A1 (Figure 2b). Pyrophosphate (PPi) ions
1
influenced the HNMR chemical shifts much more strongly
1
than orthophosphate (Pi). In contrast to the polyamines from
S.turris , short-chain amines such as spermine did not exhibit
corresponding phosphate-induced chemical shifts. The line-
widths of the signals A and B strongly exceed those of lines C,
C1, and C2 (Figure 2a).
Figure 2. H NMR spectra of polyamines from S. turris. a) Spectrum of
a solution of polyamine (pH 6.3; 3 mm). top: sodium phosphate
(35 mm), bottom: phosphate-free sample. The assignment of the sig-
nals is indicated in the formula of the repeating unit. The small signals
C1 and C2 were assigned to the methyl groups at the termini of the
molecule. Note that the samples usually contained residual amounts
of acetate from the purification procedure. b) Phosphate-induced
chemical shift (Dd(A1)) of line A1 defined as the chemical shift differ-
ence between the phosphate-containing and the phosphate-free
sample.
[
21,22]
Carr–Purcell–Meiboom–Gill (CPMG) experiments
À1
demonstrated a relaxation rate of only 6 s for lines C, C1,
À1
À1
À1
and C2, whereas T2 amounts to 36 s and 47 s for lines A
1
and B, respectively. An estimation of the methylene
H
[
23]
relaxation rate
expected for a single and globular poly-
amine molecule of 1.55 kDa molecular weight dissolved in
À1
water yields a value of about 3 s only. Although the
assumption of a globular shape for the polyamine molecules
may only be a rough approximation, the discrepancy between
the estimated value and the values observed for methylene H
independent of the phosphate concentration. According to
this model, the diffusion coefficient of the polyamine
molecules is determined by the diffusion within the droplets,
independent of the phosphate concentration, as was observed
experimentally (see above). In summary, we conclude that the
polyamines from S.turris form aggregates under the solution
conditions described. The addition of phosphate results in the
formation of larger assemblies by the attraction of such
preformed droplets. This concept could be confirmed by
1
(
lines A and B) is surprisingly large. Aggregation of the
polyamine molecules is a reasonable explanation for this
À1
observation. However, neither T2 nor the diffusion coef-
ficient were found to depend on the phosphate concentration.
We therefore assume that the polyamine molecules dissolved
in water form a microemulsion even in the absence of
phosphate ions. If so, phosphate ions could attract the
preformed droplets to build larger, loosely bound assemblies.
3
1
P NMR spectroscopy on the phosphate-containing samples
(see Supporting Information) and by dynamic light scattering
experiments performed on phosphate-free and phosphate-
containing polyamine solutions.
The viscosity within the polyamine droplets are expected to
À1
be different from that of water and determines T2
,
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Phosphate-induced assembly of polyamines appears to be
a prerequisite for silica formation, but the mechanism is
unclear. Therefore, some of the partial reactions of silica
formation were analyzed separately. First, the kinetics of
monosilicic acid polymerization in the physiologically rele-
vant pHrange was determined in the absence or presence of
S.turris polyamines. Monosilicic acid was easily determined
by gas chromatography after conversion into the correspond-
[
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2
polyamines that contain secondary amino groups.
How-
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ever, as the formation of silica nanospheres strictly depends
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itation experiment (Figure 1a, blue line) would represent the
period required for the spontaneous polymerization of
monosilicic acid. Only after the production of sufficient
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[
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uncharged molecule under acidic conditions and therefore is
unlikely to interact with polyamines. Only the much more
acidic polysilicic acids are expected to interact with poly-
amine droplets through ionic interactions as well as through
numerous hydrogen bonds with polyamine droplets. These
interactions result in polysilicic acids becoming highly con-
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These model studies define a cooperative function of
polyamines and multivalent anions in the production of silica.
In silaffins, nature has elegantly combined both structural
elements in a single molecule. Self-assembly of phosphory-
[
7]
lated silaffins has previously been documented. Notably,
nonphosphorylated silaffins are only capable of precipitating
silica in vitro if phosphate or other polyvalent anions are
added to the solutions. This behavior is in line with the
observations reported herein: Silica precipitation is only
triggered if phosphate or other polyvalent anions are added to
solutions that contain polyamines and silica precursors. At the
present time, however, we have no experimental information
about the nature of the polyvalent anion acting in vivo in the
polyamine-directed silica formation in S.turris cells.
Received: June 24, 2003
Revised: July 24, 2003 [Z52212]
Keywords: biomimetic synthesis · diatoms · nanostructures ·
.
polyamines · silica
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