Utilization of the pleiotropy of a peptidic aptamer to fabricate heterogeneous nanodot-containing multilayer nanostructures

Article abstract of DOI:10.1021/ja057262r

Peptide aptamers (=binders) against inorganic materials often show a capacity for mineralization of their target atoms; thus they are able to function both as binding molecules and as mediators for mineralization. Although the mechanisms underlying these two properties of peptide aptamers are not yet fully understood, they have been used separately to fabricate various nanostructures. Here, we present a novel method of nanofabrication, in which binding and mineralization by a peptide aptamer are alternately utilized to assemble multilayered nanostructures comprised of metal loaded cage proteins ornamented with Ti-binding peptides.

Full text of DOI:10.1021/ja057262r

Published on Web 01/18/2006
Utilization of the Pleiotropy of a Peptidic Aptamer To
Fabricate Heterogeneous Nanodot-Containing Multilayer
Nanostructures
†
‡
,†
Ken-Ichi Sano, Hiroyuki Sasaki, and Kiyotaka Shiba*
Contribution from the Department of Protein Engineering, Cancer Institute, Japanese
Foundation for Cancer Research and CREST, JST, Koto, Tokyo 135-8550, Japan, and
Department of Molecular Cell Biology, Institute of DNA Medicine, The Jikei UniVersity School
of Medicine, Minato, Tokyo 105-8461, Japan
Received October 24, 2005; E-mail: kshiba@jfcr.or.jp
Abstract: Peptide aptamers ()binders) against inorganic materials often show a capacity for mineralization
of their target atoms; thus they are able to function both as binding molecules and as mediators for
mineralization. Although the mechanisms underlying these two properties of peptide aptamers are not yet
fully understood, they have been used separately to fabricate various nanostructures. Here, we present a
novel method of nanofabrication, in which binding and mineralization by a peptide aptamer are alternately
utilized to assemble multilayered nanostructures comprised of metal loaded cage proteins ornamented
with Ti-binding peptides.
Introduction
suggesting TBP-1 recognizes a local electrostatic structure
shared among the oxidized surfaces of these three metals.
One of the most remarkable aspects of biomacromolecules
is their ability to recognize a wide array of substances with a
high degree of specificity, as exemplified by antibody-antigen
TBP-1 also possesses the capacity to mediate mineralization.
In the presence of TBP-1, nanocrystals 300-500 nm in size
11
1
were grown from silver nitrate solution. Similarly, silicification
from prehydrolyzed tetramethoxysilane (TMOS) was accelerated
in the presence of TBP-1 and was suppressed by amino acid
substitutions in TBP-1 that also impaired the binding of the
interactions. This feature of biomolecules has been extensively
explored with the aim of developing new tools in material
2
,3
sciences, and a number of artificial peptide aptamers that
specifically recognize various inorganic materials have been
1
1
mutant peptide to Ti, Ag, and Si. Thus, TBP-1 is bifunctional
in that it acts as both a metal-binding peptide and a mediator
for mineralization.
created by selecting binders from random arrays of amino acids
displayed on phages or bacteria.3-10 We previously used a
peptide-phage display system to isolate a peptide aptamer (TBP-
1
, titanium binding peptide-1) that electrostatically binds to Ti
TBP-1 comprises 12 amino acids, and its sequence is NH2-
Arg-Lys-Leu-Pro-Asp-Ala-Pro-Gly-Met-His-Thr-Trp-COOH.9
Mutational analyses have shown that it is the first hexapeptide
in the sequence that mediates Ti binding and that the identities
9
surfaces. Subsequently, we found that TBP-1 also specifically
binds to Ag and Si surfaces, but not to Au, Cr, Pt, Sn, Zn, Cu,
or Fe under conditions where nonspecific hydrophobic interac-
tions were suppressed.11 Mutations that diminished the binding
9
of Arg1, Pro4, and Asp5 are critical for such binding. Moreover,
9
,11
of TBP-1 to Ti also diminish its affinity for Ag and Si,
this hexapeptide (called minTBP-1) has the ability to endow
xenogeneic molecules with specific affinities. For instance, we
inserted a DNA fragment encoding minTBP-1 into the 5′ end
of the gene encoding the L-chain of horse ferritin, yielding a
ferritin-like cage protein, minT1-LF, with a diameter of about
12 nm in Escherichia coli that was able to bind to QCM sensors
made of Ti or SiO2, but not to one made of Au, in the presence
†
Japanese Foundation for Cancer Research and CREST.
The Jikei University School of Medicine.
‡
(
(
(
1) Amit, A. G.; Mariuzza, R. A.; Phillips, S. E.; Poljak, R. J. Science 1986,
2
33, 747-753.
2) Flynn, C. E.; Lee, S. W.; Peelle, B. R.; Belcher, A. M. Acta Mater. 2003,
1, 5867-5880.
3) Sarikaya, M.; Tamerler, C.; Jen, A. K.; Schulten, K.; Baneyx, F. Nat. Mater.
5
2
003, 2, 577-585.
1
2
(
(
4) Brown, S. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 8651-8655.
5) Kase, D.; Kulp, J. L. I.; Yudasaka, M.; Evans, J. S.; Iijima, S.; Shiba, K.
Langmuir 2004, 20, 8931-8941.
of 0.05% Tween 20. QCM analyses also showed that minT1-
LF possessed stronger affinities for Ti and SiO2 (KD ) 3.82
nM for Ti and 4.44 nM for SiO2) than the parental TBP-1 (KD
) 13.2 µM for Ti), affinities comparable to those typically
(
(
(
(
6) Lee, S. W.; Mao, C.; Flynn, C. E.; Belcher, A. M. Science 2002, 296,
8
92-895.
7) Naik, R. R.; Brott, L. L.; Clarson, S. J.; Stone, M. O. J. Nanosci.
Nanotechnol. 2002, 2, 95-100.
12
observed with natural biomolecules. This increased affinity
8) Naik, R. R.; Stringer, S. J.; Agarwal, G.; Jones, S. E.; Stone, M. O. Nat.
Mater. 2002, 1, 169-172.
almost certainly reflects the multivalency of the protein (ferritin
9) Sano, K.; Shiba, K. J. Am. Chem. Soc. 2003, 125, 14234-14235.
(
10) Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M.
Nature 2000, 405, 665-668.
(12) Sano, K.; Ajima, K.; Iwahori, K.; Yudasaka, M.; Iijima, S.; Yamashita, I.;
(
11) Sano, K.; Sasaki, H.; Shiba, K. Langmuir 2005, 21, 3090-3095.
Shiba, K. Small 2005, 1, 826-832.
10.1021/ja057262r CCC: $33.50 © 2006 American Chemical Society
J. AM. CHEM. SOC. 2006, 128, 1717-1722
9
1717

A R T I C L E S
Sano et al.
13
is a polyhedral protein composed of 24 subunits ) and the
reduced diffusion coefficient of the immobilized minTBP-1.
14
Polyhedral cage proteins such as virus capsids, lumazine
synthase,15 and heat-shock proteins, as well as ferritin,17 have
been attracting attention as promising building blocks for
nanofabrication because they can hold a variety of inorganic
materials within their interior spaces. We have already shown
that N-terminal engineered recombinant ferritin retains its ability
to store oxidized Fe within its inner space, so that a peptide
aptamer displaying ferritin could serve as a versatile block for
16
1
2
allocating inorganic nanoparticles to specific regions. Here,
we show a novel method of nanofabrication, in which binding
and mineralization by a peptide aptamer are alternately utilized
to assemble multilayered nanostructures composed of metal-
loaded peptide aptamers displaying ferritin.
Figure 1. Silicification catalyzed by minT1-LF. (A) Silicification was
started at time ) 0 by mixing prehydrolyzed 0.1 M TMOS in TBS with
0
.5 mg/mL minT1-LF (orange) or ∆1-LF (green). Silica formation was
monitored by measuring scattered light (λ ) 350 nm, arbitrary units) at
0° with a JASCO FP-6500. (B) SEM image of the silica formed in the
9
presence of minT1-LF. Bar represents 1 µm.
Experimental Section
LF in TBS. To form the first layer of minT1(R1A)-LF or ∆1-LF, we
used an Au-coated sensor. To prepare TEM samples, we used an Au-
coated sensor because the Ti-coated sensor fractured during cross
sectioning.
Ferritin Preparation. Construction, expression, and purification of
12
minT1-LF and ∆1-LF have been described previously. The expression
plasmid for minT1(R1A)-LF, in which the first Arg of minTBP-1 was
substituted with an Ala, was constructed by inserting a 5′-phosphoryl-
ated oligonucleotide duplex (KY-1439, GAT CCA TAT GGC GAA
ACT TCC GGA TGC GAG CT; and KY-1440, CGC ATC CGG AAG
TTT CGC CAT ATG) into the BamHI and SacI sites of the pKIT0
After the first layer of ferritin was formed (judged by changes of
resonance frequency of QCM), the sensor was washed with 1.5 mL of
TBS and then incubated with 0.1 M prehydrolyzed TMOS in TBS.
The silicification process was terminated by flowing 1.5 mL of TBS
into the measurement chamber. Formation of the second (and later)
ferritin layer was accomplished in the same way as the first.
EM Observations. To make observations using SEM, we fabricated
the layers on a mirror-polished Ti substrate (6 mm × 6 mm, JIS I
grade, Shinkinzoku-Kougyou, Toyonaka). Before layer formation, the
Ti substrates were extensively washed with TBS five times and then
incubated for 10 min at room temperature with 1 mL of 0.1 mg/mL
Fe@minT1. After the substrates were washed with TBS, the silicifica-
tion was started by incubating the substrate with 1 mL of 0.1 M
prehydrolyzed TMOS in TBS for 10 min. The same procedures were
repeated to obtain two alternating layers of Fe@minT1-LF and silica.
The surfaces were then coated with gold-palladium using a HITACHI
E-1030 ion sputter at 14 mA for 150 s and observed using a Hitachi
S-4300 at 5 kV.
12
plasmid. The expression and purification of minT1(R1A)-LF followed
the procedures for minT1-LF and ∆1-LF. The molecular weight of the
mutated L-chain in the assembled ferritin was determined to be 20435.2
using MALDI-TOF. This value agreed well with the calculated
molecular weight of 20426.1 if the first methionine residue was
posttranslationally removed in E. coli, as was the case for ∆1-LF. The
concentrations of the apoferritins were determined spectrophotometri-
cally with a molecular extinction coefficient of 13370 at 280 nm.
The purified apo-minT1-LF particles were filled with Fe, Co, or
12,18,19
CdSe as described
to obtain Fe@minT1-LF, Co@minT1-LF, and
CdSe@minT1-LF. The concentrations of Fe, Co, and CdSe-filled
ferritins were determined using the Bradford method with apo-minT1-
LF serving as a calibrator.
Silicification Assay. Silicification was started by mixing prehydro-
lyzed 0.1 M TMOS (Shinetsu Silicone, Tokyo) in TBS (Tris-buffered
saline, 50 mM TrisHCl pH7.5 and 150 mM NaCl) with 0.5 mg/mL
minT1-LF or ∆1-LF at ambient temperature. The prehydrolysis was
done by incubating 1 M TMOS in 1 mM HCl for 5 min at 25 °C, after
which 10 volumes of TBS was added to start the reaction. Formation
of silica was monitored by measuring scattered light (λ ) 350 nm) at
20
An EM-002BF/P-20 (TOPCON, Tokyo) TEM was used at 200 kV
to observe cross sections of multilayers. The samples were prepared
using the ion-milling method21 and stained by osmium. EDS analyses
were carried out using a NORAN system VI (Thermo Electron Corp.,
Waltham); the probe radius was 0.7 nm, and the probe current was
500 pA.
9
0° with a JASCO FP-6500.
BioLBL. Multilayered nanostructures were fabricated and monitored
Results and Discussion
on the quartz sensor of a QCM-D300 (Q-sense AB, G o¨ teborg). The
sensor had a surface area of approximately 150 mm and was equipped
Silicification Activity of minT1-LF. We began the study
described here by confirming that minT1-LF inherited the
capacity for silicification exhibited by parental TBP-1. When
we incubated minT1-LF with prehydrolyzed TMOS in a test
tube, a white precipitate appeared within 1 min. No such
precipitation was seen when ∆1-LF, which lacks the minTBP-1
peptide, or minT1(R1A)-LF, in which a critical residue (Arg1)
of minTBP-1 was substituted by Ala (see below), was incubated
under the same conditions (data not shown). Analysis of light
scattering in the solutions confirmed that the precipitate formed
(as indicated by an increase in light scattering) within 60 s in
the presence of 0.5 mg/mL minT1-LF, but the same concentra-
tion of ∆1-LF caused no increase in scattering, even after
incubating 10 min (Figure 1A). SEM revealed that the precipitate
was made up of spherical particles with diameters of 150-250
2
with a measurement cell having a volume of approximately 80 µL.
The temperature of the chamber was kept at 25.0 ( 0.05 °C, and QCM
data were collected at 14.8 MHz. We used Ti- or Au-coated sensors,
both of which were purchased from Q-sense AB. The initial minT1-
LF layer was applied to the Ti-sensor by filling the measurement cell
with 0.1 mg/mL of apo-minT1-LF or 0.2 mg/mL of metal filled-minT1-
12
(
13) Banyard, S. H.; Stammers, D. K.; Harrison, P. M. Nature 1978, 271, 282-
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84.
(
14) Douglas, T.; Young, M. Nature 1998, 393, 152-155.
(
15) Laplagne, D. A.; Zylberman, V.; Ainciart, N.; Steward, M. W.; Sciutto,
E.; Fossati, C. A.; Goldbaum, F. A. Proteins 2004, 57, 820-828.
16) McMillan, R. A.; Paavola, C. D.; Howard, J.; Chan, S. L.; Zaluzec, N. J.;
Trent, J. D. Nat. Mater. 2002, 1, 247-252.
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17) Yamashita, I. Thin Solid Films 2001, 393, 12-18.
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18) Iwahori, K.; Yoshizawa, K.; Muraoka, M.; Yamashita, I. Inorg. Chem. 2005,
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19) Tsukamoto, R.; Iwahori, K.; Muraoka, M.; Yamashita, I. Bull. Chem. Soc.
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20) Bradford, M. M. Anal. Biochem. 1976, 72, 248-254.
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1718 J. AM. CHEM. SOC.
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Biomimetic Layer-by-Layer Assembly
A R T I C L E S
Figure 2. Schematic representation of BioLBL. The first monolayer of minT1-LF is formed by the interaction between minTBP-1 and Ti (i). On top of the
layer of the minT1-LF, a silica layer is deposited via mineralization mediated by minTBP-1 (ii). The second monolayer of minT1-LF is deposited through
the interaction between minTBP-1 and silica (iii). The next silica layer is deposited on the second minT1-LF layer (iv), after which the third minT1-LF layer
is deposited (v). By using different types of minT1-LF containing different metal compounds (Fe@minT1-LF, CdSe@minT1-LF, and Co@minT1-LF),
heterogeneous multilayer nanostructures are constructed (i, iii, v).
Figure 3. Characterization of multilayer structures fabricated by BioLBL. (A) Time-dependent changes in resonance frequency (f) during QCM analysis.
Note that the values on the longitudinal axis represent the negative of ∆f. Changes associated with adsorption of minT1-LF and silicification are shown by
green and gray lines, respectively. Green and gray arrowheads represent the respective time points at which minT1-LF and TMOS were infused into the
measurement chamber. V’s represent the respective time points at which wash solution (TBS) was infused. See text for (ia)-(vi). (B-D) Surfaces of the
intermediates as observed by SEM. A first ferritin layer (B), the following silica layer (C), and the second ferritin layer (D) are shown. In this experiment,
Fe@minT1-LF was used to provide high contrast for the micrograph.
nm (Figure 1B), while EDS mapping showed both Si and O to
be present (data not shown). Thus, addition of minTBP-1
endowed ferritin with the silicification and binding character-
istics of TBP-1.
BioLBL. By taking advantage of the bifunctionality of
minT1-LF, we have been able to design a novel method for the
assembly of multilayered nanostructures, which we call BioLBL
binding of minTBP-1 to the Ti [Figure 2(i)]. Twenty-four
minTBP-1 molecules are near-symmetrically distributed on the
1
2
surface of each ferritin molecule, some of which are not
engaged in Ti binding or interaction with the adjoining ferritin
molecule and are thus free to access chemical compounds in
the solution. Consequently, addition of prehydrolyzed TMOS
results in the deposition of a silica film on the layered ferritins
by virtue of silicification mediated by minT1-LF [Figure 2(ii)].
The newly formed silica film is then the binding target of
minT1-LF, and a second minT1-LF monolayer is formed on
(biomimetic layer-by-layer assembly). The BioLBL scheme is
illustrated (Figure 2). First, a solid titanous substrate is covered
with a monolayer of minT1-LF; this is achieved via the specific
J. AM. CHEM. SOC.
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A R T I C L E S
Sano et al.
the silica [Figure 2(iii)]. This second layer of minT1-LF can
then serve as catalyst for a second deposition of silica film
[
Figure 2(iv)], which in turn serves as the target for a third
minT1-LF layer [Figure 2(v)]. This cycle of minT1-LF binding
and silicification can be repeated until the desired number of
layers is reached. Because many types of metal compounds can
be encapsulated within the interior space of ferritin,18,22-27 our
BioLBL method should enable ferritin containing a variety of
nanodots to be layered in a programmable manner.
We tested the feasibility of this new method using QCM by
monitoring changes in the resonance frequency (f) of a Ti sensor
treated as described in Figure 2. Initial infusion of minT1-LF
solution into the QCM measurement cell immediately reduced
the resonance frequency of the sensor by approximately 65 Hz;
this response plateaued within 1 min and was little affected by
subsequent infusion of a wash buffer, indicating that minT1-
LF was tightly bound to the Ti surfaces [Figure 3A(ia); note
that the longitudinal axis represents negative values of ∆f]. The
idea that the adsorbed minT1-LF formed a monolayer on the
Ti substrate is supported by the following calculation: the 65
Hz reduction in resonance frequency corresponds to an increase
2
in weight of 1.1 µg/cm on the tip (when the viscoelasticity of
adsorbed molecule was neglected), which agrees fairly well with
2
the weight of 0.9 µg/cm calculated by multiplying the number
11
2
of minT1-LF molecules adsorbed (4.8 × 10 /cm , estimated
by SEM; data not shown) by the weight of a liquid-phase
-
12
28
apoferritin molecule (1.8 × 10
µg). Furthermore, two
additional infusions of minT1-LF followed by a wash did not
change the resonance frequency of the sensor, indicating the
multiple layers of minT1-LF cannot be formed under these
conditions [Figure 3A(ib)]. After the first minT1-LF monolayer
was deposited, we infused a solution containing prehydrolyzed
TMOS, which elicited a gradual decline in resonance frequency
that was preceded by an initial rapid decrease, reflecting the
layer of silica deposited via minT1-LF-mediated mineralization
[Figure 3A(ii)]. We continued the silicification reaction for
approximately 9 min, after which it was stopped by washing
out the TMOS solution with TBS. When the cell chamber was
refilled with minT1-LF solution, the resonance frequency again
declined by 65 Hz, indicating the deposition of a second ferritin
monolayer on top of the silica layer [Figure 3A(iii)]. The two
successive cycles of TMOS and minT1-LF treatments (iv-vii)
gave essentially the same pattern of change in resonance
frequency, indicating the stepwise deposition of layers on the
QCM sensor. Within the minT1-LF layers, small particles
corresponding to ferritin were visible with SEM, whereas no
particles were observed on the silica layers (Figure 3B-D).
To confirm the importance of the minTBP-1 peptide sequence
to the BioLBL process, we conducted similar experiments with
Figure 4. Time-dependent changes in resonance frequency (f) during QCM.
(A) Layers of minT1-LF were deposited on an Au-coated sensor with
intervening silica layers. Note that the values on the longitudinal axis
represent the negative of ∆f. Changes associated with adsorption of minT1-
LF and silicification are shown by green and gray lines, respectively. Green
and gray arrowheads represent the respective time points at which minT1-
LF and TMOS were infused into the measurement chamber. V’s represent
the respective time points at which wash solution (TBS) was infused. (B)
∆
1-LF was used instead of minT1-LF. (C) The mutant minT1(R1A)-LF
was used as in (C). (D) Basal level of silicification on an Au-coated sensor.
between the sensor substrate and ferritin molecules because ∆1-
LF and minT1(R1A)-LF have no specific affinity to Ti. We
therefore used a QCM sensor coated with Au that was able to
nonspecifically adsorb proteins via hydrophobic interactions.28
We previously showed that a minT1-LF did not bind to Au
∆1-LF ferritin (recombinant wild-type horse L-ferritin) and
minT1(R1A)-LF (which displays a mutant aptamer incapable
of binding or silicification). In these experiments, we deposited
the first ferritin layer by making use of a nonspecific interaction
when the hydrophobic surface of the sensor was masked by
(
(
(
22) Douglas, T.; Stark, V. T. Inorg. Chem. 2000, 39, 1828-1830.
23) Hainfeld, J. F. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 11064-11068.
24) Mann, S.; Bannister, J. V.; Williams, R. J. J. Mol. Biol. 1986, 188, 225-
12
0
.05% Tween 20. In the absence of those blocking reagents,
2
32.
ferritin irreversibly bound to Au via hydrophobic interactions
data not shown). After the nonspecific binding of the first layer
(25) Okuda, M.; Kobayashi, Y.; Suzuki, K.; Sonoda, K.; Kondoh, T.; Wagawa,
(
A.; Kondo, A.; Yoshimura, H. Nano Lett. 2005, 5, 991-993.
(26) Wong, K. K. W.; Mann, S. AdV. Mater. 1996, 8, 928-932.
(
(
of ferritin to the sensor, we were able to fabricate multilayer
structures composed of silica and ferritin using minT1-LF as
the ferritin source (Figure 4A). When we used ∆1-LF instead
27) Yamashita, I.; Hayashi, J.; Hara, M. Chem. Lett. 2004, 33, 1158-1159.
28) H o¨ o¨ k, F.; Rodahl, M.; Brzezinski, P.; Kasemo, B. J. Colloid Interface Sci.
1
998, 208, 63-67.
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Biomimetic Layer-by-Layer Assembly
A R T I C L E S
Figure 5. Fabrication of heterogeneous multilayer structures using metal-loaded minT1-LF. (A) Time-dependent changes in resonance frequency (f) during
QCM analysis of the fabrication of heterogeneous multilayer structures. In this experiment, Fe@minT1-LF, CdSe@minT1-LF, and Co@minT1-LF were
layered on an Au-coated sensor in that order with intervening silica layers. See Figure 2 legend for details. (B) Same as (A), but the order was changed to
Fe@minT1-LF, Co@minT1-LF, and CdSe@minT1-LF. (C) TEM image of the cross-section of a multilayer structure having three Fe@minT1-LF layers.
The space between the Ti substrate and the minT1-LF layer is believed to be an artifact introduced during preparation of the sample. (D) EDS mapping of
a cross-section of the Fe@minT1-LF, CdSe@minT1-LF, and Co@minT1-LF layers. (E) EDS mapping of a cross-section of the Fe@minT1-LF, Co@minT1-
LF, and CdSe@minT1-LF layers.
of minT1-LF, however, deposition of the first silica layer
proceeded at a very slow rate that was comparable to the rate
observed with silicification of Au (Figure 4B and D). Further-
more, we were unable to apply the second ∆1-LF layer to the
silica layer (Figure 4B). Similar results were obtained with
minT1(R1A)-LF (Figure 4C), confirming that minTBP-1’s
ability to mediate both binding and mineralization is critical
for the BioLBL process.
Fabrication of Nanodot-Containing Multilayers.
The BioLBL procedure also enabled us to accumulate different
molecular layers sandwiched between layers of silica. For
instance, we prepared three types of minT1-LFs containing
different inorganic materials within their respective inner
spaces: Fe@minT1-LF contained oxidized Fe, like natural
ferritin, while Co@minT1-LF and CdSe@minT1-LF contained
oxidized Co and CdSe, respectively. Using these three minT1-
LFs, we fabricated two different multilayer structures, in which
Fe@minT1-LF, CdSe@minT1-LF, and Co@minT1-LF were
stacked in that order or, alternatively, they were stacked in the
order Fe@minT1-LF, Co@minT1-LF, and CdSe@minT1-LF.
We also fabricated the multilayer that contained three Fe@minT1-
LF layers. The QCM signal indicated that the expected layers
were formed with these metal-filled ferritins by alternately
applying silica precursors and ferritin solution (Figure 5A and
B). After three ferritin layers sandwiched by silica layers were
deposited, thin cross-sections of the structures were prepared
by ion-milling and observed using TEM. Although disturbances
most likely due to the cross-sectioning process were apparent,
the micrograms clearly showed three layers of ferritin molecules
whose thickness was approximately 30 nm (Figure 5C), and
EDS mapping further confirmed that, as expected, three types
of ferritin were layered (Figure 5D and E). Thus, by using the
BioLBL method, we were able to fabricate heterogeneous
multilayer nanostructures in a programmable manner.
Conclusions
In this study, we fused minTBP-1 with the L-subunit of
ferritin to convey the binding and mineralization capabilities
of the peptide aptamer to the cage protein. Alternative applica-
tion of these two capabilities enabled construction of multiple
layers of ferritin separated by intervening layers of silica. TEM
revealed the silica layers to be very thin and to apparently act
like the mortar between bricks, stabilizing the lamellar structure
(Figure 5C). Using this method, we were able to fabricate
structures made up of distinct ferritin layers, each containing
different nanodots of metal compounds. By fusing the aptamer
with other cage proteins having inner spaces of differing size
(e.g., 4 nm for Listeria innocua ferritin-like protein29 and 18
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A R T I C L E S
Sano et al.
14
nm for cowpea chlorotic mottle virus ), it should be possible
to fabricate stacks of nanolayers composed of arrays of
nanocrystals of distinct size on various substrates. Furthermore,
other proteinous biomolecules (antibodies, receptors, etc.) as
30
well as polymer micelles containing inorganic materials could
be endowed with binding and mineralization capabilities and
incorporated into exquisite architectures fabricated using
BioLBL (Figure 6). The current experiments only harnessed
TBP-1’s capacity for silicification; however, other peptide
aptamers also reportedly show a capacity for biomimetic
Figure 6. Schematic illustration of hetero-multilayered BioLBL ultrathin
film.
6
31
7,8,32,33
mineralization including ZnS and CoPt, among others.
By combining different aptamers, we should be able to expand
our repertoire of substances used to compose the layers. The
ability to fabricate heterogeneous multilayer nanostructures
layers could enable the development of novel types of multi-
valued memory elements, battery cells, biosensors, etc.
(
29) Allen, M.; Willits, D.; Young, M.; Douglas, T. Inorg. Chem. 2003, 42,
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300-6305.
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