bioinspired synthesis of multifunctional inorganic and bio-organic hybrid materials

MINIREVIEW

Bioinspired synthesis of multifunctional inorganic andbio-organic hybrid materialsRute Andre, Muhammad N. Tahir, Filipe Natalio and Wolfgang Tremel

Institut fur Anorganische Chemie und Analytische Chemie, Johannes Gutenberg-Universitat Mainz, Germany

Keywords

biomimetic systems; biosilica; functional

protein mimics; metal oxides;

multifunctional materials; silicatein

Correspondence

W. Tremel, Institut fur Anorganische

Chemie und Analytische Chemie, Johannes

Gutenberg-Universitat Mainz, Duesbergweg

6, D-55128 Mainz, Germany

Fax: +49 6131 39 25605

Tel: +49 6131 39 25135

E-mail: [email protected]

Website: http://www.ak-tremel.chemie.

uni-mainz.de/

(Received 2 September 2011, revised 19

March 2012, accepted 20 March 2012)

doi:10.1111/j.1742-4658.2012.08584.x

Owing to their physical and chemical properties, inorganic functional mate-

rials have tremendous impacts on key technologies such as energy genera-

tion and storage, information, medicine, and automotive engineering.

Nature, on the other hand, provides evolution-optimized processes, which

lead to multifunctional inorganic–bio-organic materials with complex struc-

tures. Their formation occurs under physiological conditions, and is gover-

ened by a combination of highly regulated biological processes and

intrinsic chemical properties. Nevertheless, insights into the molecular

mechanisms of biomineralization open up promising perspectives for bioin-

spired and biomimetic design and the development of inorganic–bio-

organic multifunctional hybrids. Therefore, biomimetic approaches may

disclose new synthetic routes under ambient conditions by integrating the

concept of gene-regulated biomineralization principles. The skeletal struc-

tures of marine sponges provide an interesting example of biosilicification

via enzymatically controlled and gene-regulated silica metabolism. Spicule

formation is initiated intracellularly by a fine-tuned genetic mechanism,

which involves silica deposition in vesicles (silicassomes) under the control

of the enzyme silicatein, which has both catalytic and templating functions.

In this review, we place an emphasis on the fabrication of biologically

inspired materials with silicatein as a biocatalyst.

Introduction

In biological mineralizing systems, the formation of

inorganic structures occurs in aqueous media at neu-

tral pH by a set of biomolecules, such as proteins and

polysaccharides. A prototypical case is provided by

marine sponges and diatoms [1,2]. The control over

mineralization achieved in biological systems has been

an inspiration for the development of new synthetic

routes to materials of technological interest [3,4].

Organisms are able to synthesize a variety of inorganic

materials (calcium carbonate, calcium phosphate,

silica, iron oxide, etc.) from simple precursors under

mild reaction conditions, resulting in highly complex

structures with several levels of hierarchy, ranging

from the nano-level to the macro-level [5,6]. These

mineralized inorganic–bio-organic composite materials

are formed either by controlled condensation in spe-

cific compartments or by regulation of the concentra-

tion of the inorganic precursors with the help of

enzymes. Diatoms [7] sponges [8] and grasses [9] pro-

vide classical examples of biosilicification processes

Abbreviations

SAM, self-assembled monolayer; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TEOS, tetraethoxysilane.

FEBS Journal 279 (2012) 1737–1749 ª 2012 The Authors Journal compilation ª 2012 FEBS 1737

whereby complex and unique 3D structures are synthe-

sized even with very low concentrations of silicon pres-

ent in the surrounding environment.

The interest in biosilicification has led to great

efforts to isolate, purify and characterize proteins and

other biomolecules, especially from diatoms and mar-

ine sponges, driving the mild synthetic route of silica

polymerization in vivo. Prominent examples include

long-chain polyamines and sillafins from diatom shells

[10–13], and silicateins from marine sponges [5,14–16].

The formation of silica in these organisms proceeds

through different reaction mechanisms: in diatoms, sil-

ica is deposited passively via electrostatic interactions,

whereas in sponges silica deposition is governed by an

enzymatic process. The formation of silica spicules in

marine sponges is of particular interest, because of

their hierarchical structures and the resulting proper-

ties; that is, the spicules have high mechanical strength

and are excellent optical waveguides. Other examples

are the hexactinellid sponge Euplectella marshalli,

whose skeletal structure is composed of elaborate

cylindrical structures with six hierarchical levels [14],

and the giant basal spicules of Monoraphis chunii (also

a hexactinellid), which can reach a length of up to 3 m

and a thickness of up to 8.5 mm [17].

Nature can easily fabricate hybrid materials under

ambient conditions; these have intricate structures and

more sophisticated combined properties than materials

synthesized in the laboratory, where conventional syn-

thetic methods involve usually high temperatures for

procedures such as thermolysis [18] or sol–gel pro-

cesses combined with subsequent calcination [19].

By emulation of the chemistry behind natural miner-

alization pathways, the known biomineralization agents

might be employed to fabricate materials with non-nat-

ural compositions and a wide spectrum of properties

[20]. Molecules such as silicateins that are involved in

biomineralization have proven to be very versatile:

besides the formation of silica and polysilsesquioxanes

[21–23], these proteins can also catalyze the formation

of diferent metal oxides such as TiO2 [24], ZrO2 [25],

CaTiO3 [26], and GaOH ⁄Ga2O3 [27]. Furthermore, the

materials formed under physiological conditions by

catalysis with silicatein often exhibit crystalline poly-

morphs that normally require high temperatures

[24,27,28] or extreme pH conditions for preparation by

classical synthetic methods [29–31]. Consequently, there

is an ever-growing need to search for new bioinspired

synthetic pathways that allow the formation of materi-

als at low processing temperatures, with a wide range

of properties and compositions and a high level of

structural complexity. In this contribution, we summa-

rize the most important advances regarding the synthe-

sis of new materials by the use of silicatein, taking

advantage of its catalytic versatility. We also emphasize

the formation of oxide thin films with a wide range of

applications by surface-bound silicatein.

Reaction mechanism of silicatein andversatility of the precursors

The isolated biomolecules involved in silica formation,

e.g. silaffins, siladicins, and silicateins, not only show an

accelerated silica polymerization from silica precursors

in vitro, but are also a source of inspiration for the use

of other biomolecules, such as synthetic polyamines,

polypeptides, and a variety of polymers that mimic the

active site of either silaffin or silicatein (e.g diblock co-

polymers) to perform similar tasks [5,11,32–36]. In fact,

these molecules in combination with site-directed muta-

genesis allow the elucidation of a plausible reaction

mechanism. The structures, compositions and molecular

masses of the bioinspired polymers, as well as the experi-

mental conditions used in the laboratory (e.g. buffer

composition and pH), have been shown to significantly

affect the kinetics of the condensation and precipitation

processes, and also the shape of the formed silica col-

loids, which can range from typical spherical shapes to

hexagons or other more complex forms [5,11,32–36].

For bioinspired silaffin-related silicification, the mecha-

nism of silica formation from solutions containing such

additives involves an electrostatic interaction between

positively charged amines and negatively charged silica

precursors, facilitating the passive condensation of silica

around the amine group. Additionally, the molecular

backbone acts as a template for the structure of the

deposited material [12,21,37–39] (for detailed informa-

tion on the chemistry of silica in biomineralized systems,

see [40]).

In contrast, to other organisms that deposit silica in

a passive, template-controlled manner [10–13,32–

36,38,39,41], marine sponges (phylum Porifera) show

the singular ability to actively synthesize their siliceous

skeleton enzymatically [5,7,14–16,21,37,42–46]; silica-

tein-a, silicatein-b, silicatein-c (subunits), silicase and

silintaphin-1 are known representatives of the proteins

responsible for sponge biosilification. These proteins

have been isolated and cloned from different siliceous

sponges (e.g. Tethya aurantium and Suberites domuncu-

la) [7,15,16,37,42–45]. Silicatein-a not only catalyzes

the formation of silica from orthosilicic acid under

in vitro conditions, but can even utilize different

related substrates to produce other metal oxides. This

indicates that silicateins have a relatively flexible active

center, and a very general mechanism can therefore be

assumed to underlie their catalytic activity.

Bioinspired synthesis of inorganic materials R. Andre et al.

1738 FEBS Journal 279 (2012) 1737–1749 ª 2012 The Authors Journal compilation ª 2012 FEBS

The charge density associated with the polarity of the

Si–O–C bonds makes metal alkoxide precursors (e.g. Si,

Ge, Sn, Ti, Zr, and Hf) susceptible to hydrolysis. How-

ever, simple alkoxides such as tetraethoxysilane (TEOS)

do not occur in nature; therefore, other molecules, such

as esters, alcohols, sugars, and catechols, may play an

active role in the transport, storage and sequestration of

silicic acid. It is known that, in both diatoms (silica

deposition vesicles) and marine sponges (silicassomes),

silica is accumulated in intracellular vesicles in higher

concentrations than in those where spontaneous poly-

merization occurs (> 100 p.p.m.) [47]. This suggests

that, parallel with the sequestration ⁄ storage mechanism,

a stabilization process preventing the spontaneous poly-

merization of silica must be active [32,48–50]. Because

of this complex silicon sequestration mechanism, the

natural substrate for silicatein has not been identified

unambiguously, although the most likely candidate

seems to be orthosilicic acid, because of its natural

abundance in seawater. Nevertheless, it has also been

speculated that the in vivo enzymatic function of silica-

tein is the activation of silanol groups (Si–OH bonds) of

silicic acid esters [5,21,46], because there is currently no

evidence to support the in vivo presence of Si–O–C con-

jugates in demosponges or their availability in nature.

Several other silicon alkoxides have been used in vitro

(as synthetic substrates) to assess the intrinsic catalytic

activity of silicatein [51,52]. Interestingly, the enzymatic

hydrolysis and polycondensation could be extended to

various nonbiogenic oxides, starting from the respective

alkoxide precursors.

Typically, the sol–gel synthesis of metal oxides from

the corresponding alkoxides proceeds in two steps: (a)

hydrolysis of a metal alkoxide to yield a metal hydrox-

ide (olation); and (b) polycondensation (oxolation)

through the condensation of two metal hydroxide spe-

cies, with concomitant release of water (oxolation), or

of a metal hydroxide with a metal alkoxide, with

release of alcohol (alkoxolation) [53], leading, ulti-

mately, to an oxide network [54,55]. Depending on the

charge density of the metal and the coordinating

strength of the ligand, the hydrolysis and the polycon-

densation reactions rates vary to a considerable extent;

they can be very fast for sterically less hindered metal

alkoxides. As a result, early transition metals of

groups 4 and 5, such as titanium, zirconium, hafnium,

and niobium, are more prone to nucleophilic attack by

water molecules, whereas silicon alkoxides are more

resistant to nucleophilic substitution by water mole-

cules. Therefore, hydrolysis and polycondensation of

silicon alkoxides must be initiated either by a catalyst

(acid, base, fluoride, or biomolecule) or elevated tem-

peratures [54]. Recombinant and native silicatein-a

were found to be active catalysts for the hydrolysis of

silicon alkoxides to yield biosilica at neutral pH and

ambient temperature [5,21,46].

Synthesis of nonbiological metaloxides with silicatein

As described above, silicatein-a possesses the ability to

catalyze the hydrolysis and condensation of various

metal oxides that do not occur in nature. This nonbio-

logical activity of silicatein can be attributed to the

similarity in size and charge of the electrophilic centers

of the substrate molecules and the steric tolerance of

the silicatein-a active center. One of the most difficult

tasks in demonstrating the catalytic activity of silica-

tein-a towards nonbiological metal oxides is the choice

of suitable precursor compounds, because most metal

alkoxides are very unstable and will spontaneously

react with water at room temperature to form an

amorphous metal oxide precipitate [53–55], precluding

their use as silicatein-a precursors. For titania, this

problem could be circumvented by using titanium

bis(ammonium lactato) dihydroxide as a water-stable,

alkoxide-like precursor [23], whereas for zirconia and

tin dioxide, the hexafluoro complexes (ZrF2�6 and

SnF2�6 ) proved to be suitable choices [56,57]. The ener-

getics imposed by the surface and bulk lattice energies

lead to the formation of metastable polymorphs whose

synthesis under standard conditions would require high

temperatures or extreme pH conditions. The mobility

of the reactant atoms at ambient temperatures prevents

the formation of well-crystallized products, yielding

nanocrystalline or partially amorphous compounds

(e.g. rutile-TiO2 and c-Ga2O3) [26,58] with reduced free

surface area, owing to particle aggregation. The forma-

tion of stabilized amorphous or nanocrystalline phases

for structural purposes is favored from a biological

perspective [10,58].

Native silicatein filaments can catalyze and template

the in vitro formation of nanocrystalline gallium oxohy-

droxide (GaOOH) and the spinel polymorph of gallium

oxide (c-Ga2O3), using stable gallium nitrate as precur-

sor [26]. Gallium (Ga3+), like its Al3+ congener, is an

example of aquo acid formation; that is, at neutral pH,

the [Ga(H2O)6]3+ hexaaquo complex predominates,

whereas in acidic or basic solution, protonation ⁄depro-tonation reactions lead to the formation of unstable

aquo ⁄hydroxo complexes such as [Ga(H2O)6 )

n(OH)n](3 ) n)+ [48–50,59,60]. Consequently, in acidic

media, the condensation of hydrolyzed Ga3+ will pro-

ceed via either olation or oxolation [53].

In contrast, under neutral pH conditions or in the

presence of thermally denatured protein filaments, no

R. Andre et al. Bioinspired synthesis of inorganic materials


hydrolysis of [Ga(H2O)6]3+ occurred. However, upon

incubation of gallium nitrate with protein filaments,

nanocrystalline GaOOH was formed [60]. Moreover,

when lower Ga3+ concentrations were present, silica-

tein-mediated hydrolysis of gallium nitrate yielded

nanocrystalline spinel-type gallium oxide (c-Ga2O3) as

the kinetically preferred product. Electron diffraction

analysis showed that, in both cases, the nanocrystals

formed via the silicatein-mediated route exhibit a pre-

ferred orientation relative to the axis of the main pro-

tein filament. This suggests that, in the case of

c-Ga2O3, the protein filaments are responsible not only

for catalyzing the hydrolysis of the water-stable gal-

lium nitrate precursor ([Ga(H2O)6]3+) and inducing

the crystallization of the spinel oxide, but also for

influencing the preferred pseudo-oriented crystal

growth relative to the protein.

From a materials science perspective, perovskites

with a generic structure ABO3 drawn from a range of

metals, subject to certain size constraints, represent a

very flexible system. The range of possible cationic

substitutions is limited only by constraints on thermo-

dynamic stability, as represented in terms of the

Goldschmidt factor. The perovskite structure can toler-

ate significant nonstoichiometry and partial substitu-

tion. Its compositional variability is linked directly to

its physical properties, including ferroelectric, dielec-

tric, pyroelectric, piezoelectric, ferromagnetic ⁄ ferrimag-

netic, electrically (super)conducting or catalytic

behavior [50,61]. The different physical properties of

perovskite-phase materials are related to their phase

transitions, which in turn are sensitive to variables

such as chemical composition, purity, numbers of sur-

face and bulk defects, grain size, and sintering condi-

tions. Hence, the control of these parameters is critical

for effective property control. The methods for synthe-

sizing perovskites with an ABO3 generic structure are

far from the physiological conditions. However,

sponge filaments isolated from T. aurantium are able

to template barium titanium oxyfluoride (BaTiOF4) as

dispersed florets of nanocrystals [62]. Unlike previous

silicatein-catalyzed reactions involving the formation

of silica, titania, or gallium oxide, the synthesis of

BaTiOF4 required a cofactor, H3BO3, which was pro-

posed to scavenge excess fluoride ions generated by the

hydrolysis of the BaTiF6 precursor [58].

Deposition of thin films withsurface-bound recombinant silicatein

For many applications, it is necessary to synthesize

thin films of metal oxides on solid surfaces rather than

in solution, with simultaneous control of the pattern

and shape of the deposited inorganic materials over

more than the micrometer scale. However, the chemi-

cally driven deposition of uniform silica coatings on

solid surfaces has not been achieved under ambient

conditions, despite many potential applications for

such materials in sensors [63], membranes, and struc-

tural materials [64]. With, again, nature as inspiration,

metal oxide thin films on solid supports could be pre-

pared by immobilizing silicatein-a and making use of

its catalytic properties. Moreover, the creation of silica

patterned thin films by the use of immobilized poly-

peptides and polyamines that act as templates has been

reported with several methods, such as electrostatic

deposition [65,66], direct write assembly [67], holo-

graphic patterning [68], photolithography [67], and sur-

face-initiated polymerization [69]. However, this

approach is beyond the scope of this review.

Silicatein-a was immobilized on various inorganic

substrates with His-tags, Glu-tags, and Cys-tags.

Initially, the immobilization and activity of surface-

bound recombinant silicatein-a containing a His-tag

(His6 additional sequence) was demonstrated on solid

supports by the formation of heterogeneous silica, tita-

nia and zirconia films [60,70,71]. In the initial experi-

ments, silicatein-a was immobilized on self-assembled

monolayers (SAMs) on Au(111) surfaces by use of a ni-

trilotriacetic acid-terminated organic thiol (Fig. 1A).

The thiol binds to the gold surface, and the nitrilotri-

acetic acid terminus remains free for Ni2+ and His-tag

protein complexation [72]. The kinetics of the SAM

deposition of the nitrilotriacetic acid organic thiol, the

subsequent chelation of Ni2+ and the anchoring of sili-

catein were monitored by surface plasmon resonance

spectroscopy and atomic force microscopy. The hydro-

lytic activity of surface-bound silicatein-a was con-

firmed by the formation of a thin layer of silica (SiO2,

surface coverage � 70% with TEOS as a metal alkox-

ide precursor; Fig. 1B–D). The nitrilotriacetic acid–

Ni2+ linker group selectively binds to the His-tag of

the recombinant protein, thereby providing a controlled

spatial orientation. These factors (linker and well-

defined spatial orientation) are of major importance for

the catalytic activity of a surface-bound enzyme, which

is constrained by: (a) the accessibility of the active site

of the enzyme to the substrate; and (b) protein folding

(e.g. denaturation). In an alternative biomimetic silicifi-

cation approach – based on kinetically controlled cata-

lytic hydrolysis and polycondensation – synthetic

analogs of the active site of the enzyme were immobi-

lized on surfaces. Therefore, attempts were made to

anchor recombinant silicatein-a (His-tagged) on gold

surfaces with a reactive ester polymer [59] (Fig. 1E).

After protein complexation, the film thickness increased



by 3.4 nm, which is close to the theoretical diameter of

the 24-kDa silicatein-a. This was confirmed by a posi-

tive cross-reaction between polyclonal antibodies raised

against silicatein-a and the surface, which was observed

by confocal laser microscopy (Fig. 1F). The catalytic

activity was verified by the formation of 50–60-nm lay-

ered particles of TiO2 and a thin film of cubic ZrO2 (an

unusual ZrO2 polymorph, in particular at ambient

reaction conditions) on surface-bound silicatein-a, withtitanium bis(ammonium lactato) dihydroxide and hexa-

fluoro-zirconate (ZrF2�6 ) as stable nonbiological precur-

sors (Fig. 1G,H).

Tin dioxide (SnO2) is a well-studied semiconductor.

Of particular interest are SnO2-coated glass surfaces,

A

E

Nitrilotriacetic acidNitrilotriacetic acid Nitrilotriacetic acid

Nitrilotriacetic acidNitrilotriacetic acid

F G H

B C D

Fig. 1. Biocatalytic activity of surface-bound silicatein-a immobilized with nitrilotriacetic acid-based ligands. (A) Chemical structure of nitrilotri-

acetic acid-terminated alkanethiol used as a SAM to immobilize His-tagged silicatein-a to gold (111) surfaces. (B–D) SEM images of silicatiza-

tion onto surfaces functionalized with nitrilotriacetic acid alkanethiol without Ni2+ (A) and nitrilotriacetic acid alkanethiol with Ni2+ chelating

silicatein (B, C). No observable formation of SiO2 occurred on nitrilotriacetic acid alkanethiol-modified surfaces (A). In contast, Ni2+-chelated

silicatein immobilization onto nitrilotriacetic acid alkanethiol surfaces induced the formation of SiO2. (E) Schematic representation of the

immobilization of His-tagged silicatein-a via a SAM. Silicatein-a was immobilized by tailoring the Au(111) surface by using: (a) cysteamine

SAMs; (b) reactive ester polymer that was covalently bound to the amine head group of cysteamine; (c) and nitrilotriacetic acid molecules

that were further immobilized by using the remaining reactive ester group present in the backbone of the polymer. (F) Confocal laser scan-

ning microscopy images of immunodetection of immobilized silicatein-a with polyclonal antibodies raised against silicatein-a (PoAb-SILA).

(G, H) High-resolution SEM images of TiO2 and ZrO2 formed by catalysis with surface-bound silicatein.



because they form the basis for transparent semicon-

ductors. These SnO2-coated surfaces were obtained by

immobilizing silicatein-a (His-tagged) on glass, as con-

firmed by a positive cross-reaction with antibodies

(Fig. 2A). The catalytic activity was demonstrated by

the formation of a dense film of SnO2, by use of a

water-stable tin precursor (Na2SnF6), with surface-

bound silicatein-a at neutral pH and room temperature

[57] (Fig. 2B). Scanning electron microscopy (SEM)

analysis of the surface showed that the film was com-

posed of spherical SnO2 agglomerates with an average

size of 50 nm that, in turn, were composed of smaller

particles (between 2 and 5 nm) (Fig. 2C). This mor-

phology of the product can be rationalized as arising

from a templating effect of the protein agglomerates

[43,57]. Crystallographic analysis of the nanodomains

showed the presence of cassiterite-type SnO2, the poly-

morph selection being independent of the reaction

parameters. At the macroscopic level, the transparency

of the glass slides was maintained after catalytic depo-

sition of SnO2 (both for visible and for UV light),

making both functionalization and surface coating a

facile approach for the production of new materials

with better performance (Fig. 2D,E).

In a similar manner, silica-coated magnetic nanopar-

ticles c-Fe2O3@SiO2 could be fabricated [73]. For this

purpose, His-tagged silicatein-a was immobilized on

c-Fe2O3 nanoparticles functionalized with a nitrilotri-

acetic acid-containing polymeric ligand and by making

use of the efficient chelating properties of Ni2+. The

particle-bound silicatein-a was shown to be active for

catalyzing and structurally directing the deposition of

a protective biosilica shell around the magnetic oxide

nanoparticles.

This convenient surface modification strategy based

on a multifunctional nitrilotriacetic acid-containing

polymeric ligand could be generalized. His-tagged silica-

tein-a was immobilized on the highly hydrophobic sur-

face of WS2 nanotubes, which – in turn – made WS2highly water-soluble (Fig. 3A) [74]. The protein binding

was confirmed by scanning force microscopy. The activ-

ity of WS2 surface-bound silicatein-a was demonstrated

by the formation of a dense and hydrophilic TiO2 coat-

ing on hydrophobic WS2 surfaces (Fig. 3B). High-reso-

lution transmission electron microscopy (TEM) images

showed nanocrystalline domains with a fringe spacing

of 0.32 nm, which is close to the (110) lattice spacing of

rutile-type TiO2 (Fig. 3C).

A B C

D E

Fig. 2. Enzymatic formation of SnO2 by silicatein-a immobilized on glass surfaces. (A) Structural and schematic representation of functional-

ization of glass slides with His-tagged silicatein-a. The surfaces were first treated with a epoxide-terminated silicane, which reacted further

with amine-terminated nitrilotriacetic acid, allowing binding of silicatein-a through Ni2+ complexation. (B) SEM overview image of the homo-

geneous deposition of SnO2. (C) Higher-resolution SEM image, showing the sphere-like SnO2 particles with average size of 50 nm. (D) UV–

visible transmittance of glass slides before (dashed line) and after (bold line) SnO2 functionalization. (E) Optical images of glass slides before

and after silicatein ⁄ SnO2 functionalization. Almost no color change was detected after functionalization with protein and SnO2 deposition,

and the glass slides remained transparent in the visible range.



Whereas most of the above examples rely on the

hydrolytic abilities of silicatein-a, its (nonphysiological)reductive properties can be utilized for the fabrication

of unusual structured composites. This was demon-

strated by biofunctionalizing TiO2 nanowires [75], first

with a bifunctional polymeric ligand containing pen-

dant catechol moieties (specific anchor groups for

metal oxides) and nitrilotriacetic acid functionalities

(complexation with Ni2+), and then with recombinant

His-tagged silicatein-a, which allowed the subsequent

silicatein-mediated growth of gold nanocrystallites on

the TiO2 surface with AuCl4– as precursor [71,76]

(Fig. 3D). The reductive properties of TiO2-supported

recombinant silicatein-a were attributed to free thiols

present in the protein [17]. Moreover, the nanocrystals

possess an unusual S3 symmetry axis, which suggests

chiral induction from the protein to the triangular-

shaped nanocrystals (Fig. 3E,F).

In order to expand the affinity of silicatein-a to

other solid supports and to simultaneously reduce the

number of reaction steps, new tags were introduced

during protein expression. For example, silicatein-acontaining a Glu-tag (Glu8) in its C-terminus was

developed, allowing specific immobilization on

hydroxyapatite [77]. Comparative studies of the

strengths of adhesion of silicatein-a and Glu-tagged

silicatein-a to hydroxyapatite showed that the addi-

tional Glu-rich sequence is, indeed, required for a

strong binding affinity ⁄ interaction. Its potential appli-

cation in dentistry was explored; that is, the treatment

of dentin tubules with Glu-tagged silicatein-a and

exposure to a silica source (sodium metasilicate was

used, owing to its negligible toxicity) led to tubule

occlusion, suggesting that this integration of protein

into toothpastes can reduce significantly hypersensitiv-

ity. The modes of binding of Glu functional groups

to calcium surfaces can be extrapolated to metal oxi-

des in general (Fig. 4A). Here Glu-tagged silicatein-awas used to functionalize TiO2 nanowires, thereby

avoiding the other additional functionalization steps

required for protein immobilization. The protein

could be detected with antibodies raised against silica-

tein-a, and its activity in the formation of SiO2 and

ZrO2 was confirmed, allowing the formation of core-

shell structures [78] (Fig. 4E,F). It may be expected

that this method can be generalized for other metal

oxide surfaces, and the possible development of other

material-specific tags would allow the synthesis of a

wide range of nanocomposite materials under physio-

logical conditions.

A B C

D E F

Fig. 3. Immobilization of silicatein on different nanostructured surfaces. (A) Digital photographs of WS2 nanotube dispersions before (left)

and after (right) functionalization with silicatein-a, showing the change in hydrophilicity of the functionalized material. (B) SEM image showing

the deposition of TiO2 in the WS2 nanotubes, catalyzed by silicatein-a. (C) High-resolution TEM image showing the interface between the

WS2 nanotube surface and the newly formed TiO2 layer, where crystalline domains are visible, corresponding to the rutile phase of titania.

(D) Schematic representation of polymer ⁄ silicatein-a-functionalized TiO2 nanowires with deposited gold nanoparticles. (E) TEM overview of

TiO2 nanowires covered with gold nanocrystallites, catalyzed by silicatein-a. (F) High-resolution TEM image of the gold nanocrystals.



Complex mineralization matrixes –silicatein interactors

The axial filament of spicules is composed of several

different biomolecules with different properties and

functions (see acompaning minireviews for more

detailed information). Silicateins are the main proteins

responsible for silica polymerization, but, recently,

another group of proteins – silintaphins – were discov-

ered that interact strongly with silicatein. It was shown

that both proteins could be colocalized in the spicule

filaments and – when incubated together – they tended

to assemble in a stoichiometric manner and to form fil-

amentous aggregates [77,79]. The role of silintaphin-1

in the activity of silicatein was also explored. For pro-

teins present in a 4 : 1 ratio (silicatein ⁄ silintaphin),maximum polymerization of SiO2 from TEOS was

observed. The use of silintaphin-1 was shown to affect

assembly of the synthetic materials. For example, when

c-Fe2O3 nanoparticles functionalized with polymeric

ligand carrying silicatein-a on its surface were coincu-

bated with silintaphin-1, needle-like spicules with

lengths of up to hundreds of nanometers, with clear-

cut edges, were formed by the assembly of the nano-

particles [77]. The interaction between the proteins was

also confirmed by exposing silintaphin-1 to TiO2 nano-

wires functionalized with silicatein-a. Both proteins

could be colocalized by immunostaining, and the

organic matrix proved to remain active for the forma-

tion of SiO2 and ZrO2 (Fig. 4B–D). It was observed

that, in the presence of both proteins, higher amounts

of SiO2 and ZrO2 were deposited, resulting in a thicker

layer than obtained with nanowire surfaces carrying

only silicatein [78] (Fig. 4E–H). This phenomenon

points to the importance of complex matrices in bio-

mineralization mimetics, where increasing the complex-

ity of the organic matrix can lead to fine-tuning of the

produced materials.

Silicatein synthesis ofpolysesquioxanes

Polysilsesquioxanes (RSiO1.5)n are organic silica ana-

logs in which each silicon center in the network pos-

sesses at least one Si–C bond [80]. Silicones have

found a wide range of commercial and technological

applications. Polysilsesquioxanes are formed by hydro-

lytic condensation of organosilane precursors (RSiX3,

where R = an organic functionality and X = alkox-

ide, amide, halide, etc.) under acidic or basic condi-

tions, and often at elevated temperatures [81].

Silicatein-a can catalyze the in vitro condensation of

A

B C D

E G

F H

Fig. 4. Glu-tagged silicatein-a immobilization on metal oxide surfaces and its interaction with silintaphin-1. (A) Schematic representation of

the possible modes of binding of the Glu domains to TiO2 surfaces, or other metal oxides in general. The Glu-tag in the recombinant silica-

tein-a consists of a Glu8 domain in the C-terminal region of the protein. (B–D) Atomic force microscopy phase-contrast images of (B) bare

TiO2 nanowires, (C) Glu-tagged silicatein-a and (D) Glu-tagged silicatein-a ⁄ silintaphin-1-functionalized TiO2 nanowires. It is possible to observe

that: (a) the Glu-tagged silicatein-a binds directly to the metal oxide surface; and (b) the increase of organic matrix is evident when both pro-

teins are incubated together, indicating their strong affinity. (E–H) Formation of SiO2 (E, G) or ZrO2 (F, H) by Glu-tagged silicatein-a (E, F) and

by Glu-tagged silicatein-a ⁄ silintaphin-1 (G, H). An evident increase in the formation of SiO2 or ZrO2 is observed when both proteins are on

the surface of TiO2 nanowires.



alkoxysilanes during a phase transfer reaction at neu-

tral pH and ambient temperature to yield silicones

[5,21,46], as confirmed by 29Si-NMR analysis. In the

absence of silicatein-a, the alkoxysilane monomer pre-

cursor was hydrolyzed only to a negligible extent.

Highly substituted precursor compounds (e.g. pheny-

lated alkoxides) blocked the polymerization reaction

completely.

Although these substrates (e.g. polysilsesquioxanes)

possessing Si–C bonds are nonbiological substrates for

silicatein-a that do not occur in nature, the Si–C bonds

do not influence the silicatein-catalyzed formation of

polysilsesquioxanes. As shown by 29Si magic angle

spinning NMR spectroscopy, only silicon centers car-

rying organic groups with varying degrees of steric

crowding and electron withdrawing and donating

properties (e.g. phenyltriethoxysilane and methyltrieth-

oxysilane) seem to affect the catalysis. More recently,

other functional polysilsesquioxanes have been pro-

duced, with silicatein-a as catalyst, further demonstrat-

ing the tolerance of the enzyme for nonbiogenic

precursors.

Organic substrates for silicatein-a

Interestingly, the catalytic activity of silicatein-a with

respect to hydrolytic ⁄polymerization reactions is not

restricted to inorganic oxides. It was extended for

organic polymers as well, by the generation of

poly(lactic acid) from a cyclic precursor (l-lactide)

through a biocatalytically controlled ring opening

polymerization mechanism. The enzyme acts a catalyst

rather than a ring opening polymerization initiator,

and was found to be adsorbed to the surfaces of the

axial filaments rather than covalently bound [82].

Synthetic analogs of silicatein-a

One of the limitations of the protein-catalyzed syn-

thetic systems is that – in general – only low concen-

trations of the proteins are available from natural

sources (e.g. spicules) or as products of laboratory

genetic manipulation [21,46]. However, in recent years,

numerous strategies have been introduced to mimic

bioinspired catalysts, such as silicatein from sponges

and silaffins from diatoms. Organic templates such as

polymers [83,84], polymer–peptide hybrids [85,86],

diblock copolypeptides [27], self-assembling peptides

[87–91] and cationic peptide amphiphiles [92] were

used to promote sol–gel condensation of silica and

other inorganic precursors. Template sol–gel processes

yield hybrid materials with a wide range of different

morphologies that are strongly influenced by factors

such as temperature, concentration, and the pH of the

reaction medium [93].

The successful application of polymeric and surface-

functionalized silicatein-a mimics had indicated that

even simple bifunctional compounds might act as func-

tional analogs of the silicatein-a active site for hydro-

lytic catalysis, as confirmed by use of a series of small

molecules with a nucleophilic terminus (e.g. –OH,

–SH, and –SC2H5) and a hydrogen bond acceptor (e.g.

a primary or tertiary amine) at the other end [94]. In

the absence of catalyst, TEOS mixed with the buffered

aqueous solution remained stable for several days.

Cysteamine (followed by ethanolamine) promoted the

highest yield of silica condensation. This result is con-

sistent with previous results obtained with synthetic

block copolypeptides and with the observation that

proteases possessing Cys residues in the catalytic triad

are enzymatically more active than proteases that con-

tain Ser residues instead [14,88].

These nucleophilic and basic functionalities (which

influence the catalytic formation of silica) are not

required to be part of a single molecule or a polymer;

they may be different molecules functionalized ⁄ chemi-

sorbed separately onto different active surfaces. This

was demonstrated for a synthetic system where an

appropriate nucleophilic function (e.g. hydroxyl) and

nitrogen bases (e.g. imidazole) were immobilized onto

two populations of gold nanoparticles (as carriers) via

self-assembled monolayers of x-functionalized organic

thiols [95]. The mechanistic assumption of the nano-

particle-bound system is that, when two nanoparticles

with different functionalities come close enough to

allow hydrogen bonding (e.g. 2–3 A), an active catalyst

is formed. This would correspond to the interaction of

a hydroxyl group from Ser and the imidazole group

from His (separation � 2 A), which is essential for

promoting silica alkoxide hydrolysis, i.e. for cleaving

the silicic ester bonds. Little or no hydrolysis occurred

when: (a) only one type of functional nanoparticle

were present; (b) either class of functional nanoparticle

was replaced with a noninteracting molecule; or (c) un-

functionalized gold nanoparticles were used.

The awareness that silica can be catalyzed efficiently

by cysteamine led to useful, convenient and low-cost

encapsulation of biological materials, such as enzymes,

antibodies, and cells, that otherwise might be damaged

by exposure to the acid, base, or heat [96]. This was

shown elegantly by encapsulating blue fluorescent pro-

tein and live Escherichia coli cells expressing green

fluorescent protein with cysteamine in 2D micropat-

terned matrices. The 2D micropatterned matrices

(obtained by microcontact printing of live cells

expressing green fluorescent protein or a solution of



blue fluorescent protein mixed with the silica precursor

and cysteamine) displayed stable and active fluores-

cence, confirming that the encapsulation with cyste-

amine was successful in maintaining the activity of the

biological materials, and that this method could there-

fore potentially be extended to the encapsulation and

micropatterning of a whole host of live cells, enzymes,

antibodies, receptors, and fluorescent and other func-

tional proteins [97].

Conclusion

The discovery that silicatein-a from demosponges can

act as a hydrolytic enzyme and – in the form of natu-

ral filaments – as a template for biosilicification has

inspired the development of new synthetic methods for

bioinspired material synthesis. The key aspects are:

(a) the kinetically controlled, catalytic hydrolysis of

molecular precursors; and (b) the templated polycon-

densation and growth of metal oxides on the surface

of the silicatein filament. The steric tolerance of the

active site of silicatein allows translation of the basic

chemical principles of silicatein-mediated catalysis and

growth to a range of nonbiological chemical substrates

that are not found in the biosphere. This review has

highlighted some examples of silicatein-inspired, low-

temperature fabrication of materials and some bioin-

spired methods that do not even require biocatalysts

or organic templates for making a wide range of

advanced nanostructured and microstructured

materials.

The next generation of methods for the fabrication

of bioinspired materials must begin to draw inspiration

from complex biological systems in which the con-

certed action of several components produces sol-

ids ⁄biomaterials, which, of course, must increase the

complexity of the synthetic analog. If this can be

accomplished, a higher degree of structural complexity

and precision may be possible. This may involve the

use of genetically manipulated proteins that are capa-

ble of building complex 2D or 3D structures via bot-

tom-up processes. Cloned biomolecules acting as

mineralization templates, e.g. spider silk proteins and

their mutants, could be employed for 3D assembly of

nanofibers, or film and foam formation. Existing

cloned peptides and proteins could be genetically opti-

mized for building up such 3D structures.

Acknowledgements

This work was supported by grants from the Bundes-

ministerium fur Bildung und Forschung Germany

(project ‘Center of Excellence BIOTECmarin’), the

Deutsche Forschungsgemeinschaft, and the European

Commission (Biomintec).

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