Large centimeter-sized macroporous ferritin gels as versatile nanoreactors

Article abstract of DOI:10.1021/cm403240j

Organized assemblies of bionanoparticles such as ferritin provides templates that can be exploited for nanotechnological applications. Organization of ferritin into well-defined three-dimensional assemblies is challenging and has attracted considerable attention recently. We have synthesized, for the first time, large (centimeter-sized) self-standing macroporous scaffold monoliths from ferritin bionanoparticles, using dynamic templating of surfactant H₁ domains. These scaffolds comprise three-dimensionally connected strands of ferritin, organized as a porous gel with porosity ～55 μm. The iron oxide inside the ferritin scaffold can be easily replaced with catalytically active monodisperse zerovalent transition metal nanoparticles using a very simple protocol. Since the ferritin is cross-linked in the scaffold, it is significantly robust with enhanced thermal stability and better tolerance toward several organic solvents in comparison to the native ferritin bionanoparticle. In addition, the scaffold macropores facilitate substrate and reagent transport and hence the monoliths containing active Pd or iron oxide nanoparticles inside apo-ferritin bionanoparticles were used as a recyclable heterogeneous catalyst for the oxidation of 2,3,6-trimethyl phenol to 2,3,6-trimethyl-1,4-benzoquinone (precursor for Vitamin E synthesis) and for Suzuki-Miyaura cross-coupling reaction in both aqueous and organic solvents. The protein shell around the nanoparticles protects them from agglomeration, a phenomenon that otherwise plagues nanoparticles-based catalysis. The presence of macropores allow the ferritin scaffold to act as catalytic monolith for continuous flow reactions having rapid reaction rates, while offering a low pressure drop. Finally, the Pd@apo-ferritin scaffold was immobilized inside a steel cartridge and used for the continuous flow hydrogenation of alkenes to their corresponding alkanes for 15 cycles without any loss of activity.

Full text of DOI:10.1021/cm403240j

Article
pubs.acs.org/cm
Large Centimeter-Sized Macroporous Ferritin Gels as Versatile
Nanoreactors
†
†
,‡
,†
Sushma Kumari, Amol Kulkarni, Guruswamy Kumaraswamy,* and Sayam Sen Gupta*
†
‡
CReST Chemical Engineering Division and Polymer Science and Engineering Division, National Chemical Laboratory,
Pune-411008, India
*
S Supporting Information
ABSTRACT: Organized assemblies of bionanoparticles such as ferritin provides templates
that can be exploited for nanotechnological applications. Organization of ferritin into well-
defined three-dimensional assemblies is challenging and has attracted considerable
attention recently. We have synthesized, for the first time, large (centimeter-sized) self-
standing macroporous scaffold monoliths from ferritin bionanoparticles, using dynamic
templating of surfactant H1 domains. These scaffolds comprise three-dimensionally
connected strands of ferritin, organized as a porous gel with porosity ∼55 μm. The iron
oxide inside the ferritin scaffold can be easily replaced with catalytically active
monodisperse zerovalent transition metal nanoparticles using a very simple protocol.
Since the ferritin is cross-linked in the scaffold, it is significantly robust with enhanced
thermal stability and better tolerance toward several organic solvents in comparison to the
native ferritin bionanoparticle. In addition, the scaffold macropores facilitate substrate and
reagent transport and hence the monoliths containing active Pd or iron oxide nanoparticles
inside apo-ferritin bionanoparticles were used as a recyclable heterogeneous catalyst for the
oxidation of 2,3,6-trimethyl phenol to 2,3,6-trimethyl-1,4-benzoquinone (precursor for Vitamin E synthesis) and for Suzuki−
Miyaura cross-coupling reaction in both aqueous and organic solvents. The protein shell around the nanoparticles protects them
from agglomeration, a phenomenon that otherwise plagues nanoparticles-based catalysis. The presence of macropores allow the
ferritin scaffold to act as catalytic monolith for continuous flow reactions having rapid reaction rates, while offering a low pressure
drop. Finally, the Pd@apo-ferritin scaffold was immobilized inside a steel cartridge and used for the continuous flow
hydrogenation of alkenes to their corresponding alkanes for 15 cycles without any loss of activity.
KEYWORDS: self-assembly, ferritin, gel, macroporous materials, catalysis, continuous flow
INTRODUCTION
applications, and ferritin has proven popular in these endeavors
due to its ready availability on large scale and the ease of its
interior mineralization. Metal ion mediated self-assembled one-
dimensional nanostrands of ferritin have shown applications as
■
In the last several years, bionanoparticles such as the iron
storage protein ferritin and the tobacco mosaic virus have
emerged as versatile building blocks for the formation of
11
1
−3
nanofiltration membranes for ultrafast water permeation.
Ferritin readily assembles into two-dimensional lattices at the
functional nanostructured assemblies.
The nanoparticles
have a layered structure comprised of a self-assembled protein
shell surrounding a core that can contain nucleic acid, protein,
and inorganic material. Recent advances in protein engineering
12
13
air−water interface and on solid substrates. Ferritin-
polymer conjugates, including ferritin, has also been assembled
in oil-in-water (o/w) and water-in-oil (w/o) pickering
4
5
allow incorporation of enzymes, transition metal catalysts,
6
,7
1
4
and zerovalent late transition metal nanoparticles into these
structures. Such modified bionanoparticles are well suited to
perform chemical transformations due to the presence of the
catalytically active core while the protein shell modulates the
emulsions to form capsulelike structures. Well-defined
three-dimensional architectures are more challenging and
have attracted considerable recent attention. The first report
of chemically cross-linked clusters of ferritin in solution
employed biotin−streptavidin linkages to create colloidal
5
−7
entrance and egress of substrate and products.
The iron-storage protein ferritin represents one of the
smallest but most practical of these nanoparticles. Its 24 protein
subunits self-assemble to form a 12 nm diameter cage with an
interior cavity of 8 nm diameter containing a ≈5 nm iron oxide
15
aggregates. More recently, composite materials containing
16−18
ferritin have been shown to have interesting properties.
Electrospun fibers of polyvinyl alcohol-ferritin composites form
19
8
nanofibrous hydrogels, that have been used as bioactuators.
core. Ferritin has been used for catalysis, biosensing, the
template synthesis of nanomaterials, and biomedical applica-
5
,9,10
tions.
Received: October 3, 2013
Revised: November 14, 2013
Published: November 14, 2013
Organized higher-order assemblies of nanoparticles provide
templates that can be exploited for nanotechnological
©
2013 American Chemical Society
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Vinylated ferritin has also been copolymerized with acrylamide
to form a hydrogel.
buffer was added and the N
before the scaffold was taken in 500 mL of 0.15 M NaCl and dialyzed
against saline for a day in the cold room.
Synthesis of Palladium Nanoparticle Encapsulated Apo-ferritin
Scaffold (Pd@apo-ferritin). An apo-ferritin scaffold was incubated
with 5 mL of 0.1 M NaCl/H O and then the pH was adjusted to 8.5
with 0.01 N NaOH, followed by the addition of aliquots of 500 μL of
2
purge was continued for another hour
20
The formation of large monolith gels from biomolecular
nanoparticles has not been reported till date. Such materials
would combine the advantages of cage-like nanoparticles (the
ability to sequester a variety of cargo in monodisperse and
robust protein cages), with those afforded by a highly porous
three-dimensional architecture, facilitating transport of sub-
strate and products to and from the functional cages. An
obvious application for such macroporous, three-dimensional
materials is continuous flow biocatalytic chemistry.
2
3
0 mM K PdCl solution for 2 h. Subsequently, the scaffold was
2 4
washed with water, followed by immediate reduction with NaBH4
solution (100 μmol in water) and incubated for an additional 2 h.
Finally, the scaffold was extensively washed with deionized water
under sonication several times to remove adsorbed Pd nanoparticles.
General Procedure for Oxidation of 2,3,6-Trimethylphenol to
Trimethyl-1,4-benzoquinone using Ferritin Scaffold. The oxidation
of 2,3,6 trimethylphenol was performed in a sealed tube containing 1
mg of ferritin scaffold in 1 mL of MeCN at 70 °C for 24 h. The
reaction was initiated by adding 0.14 mmol of H O to the reaction
mixture containing 0.05 mmol TMP. After completion of the reaction,
the amount of TMP consumed and product formed was estimated by
GC and GC-MS. The scaffold was reused for next cycle after pipetting
out the reaction mixture.
We have recently demonstrated a general route to the
preparation of macroscopic scaffolds by dynamic templating of
2
1,22
surfactant hexagonal phase (H ) domains.
On cooling
particle dispersions in 1:1 mixtures of nonionic surfactant C E
2 9
1
1
2
2
and water to below the isotropic-H1 transition temperature
(THI ≈ 44 °C), we form networks of particles that are jammed
at the H1 domain interfaces. Cross-linking the particles
followed by removal of surfactant template by extensive water
washing results in the formation of macroporous scaffolds. We
have previously demonstrated that macropore dimensions can
be varied over 2 orders of magnitude and that pores can be
readily oriented by gentle shearing. Thus, dynamic templating
is a powerful technique to produce macroporous scaffolds of a
variety of nanoparticles.
General Procedure for Suzuki−Miyaura Cross-Coupling Reaction
Using Pd@apo-ferritin Scaffold. Suzuki−Miyaura cross-coupling
reaction was investigated with Pd@apo-ferritin scaffoldat 75 °C for
1
8 h in water. The reaction was performed in an aqueous solution (1
mL), which contained aryl halide (0.023 mmol), phenylboronic acid
0.048 mmol), NaOH (0.092 mmol) as a base and Pd@apoferritin
(
scaffold (3 mg) as a catalyst. The reaction mixture was pipetted out to
a separate scaffold, and the product was extracted with chloroform.
The amount of starting materials consumed and product formed was
estimated by GC and GC-MS. Scaffold was again incubated with fresh
batch of reactants to carry out recyclability of the scaffold.
Oxidation of TMB Using Ferritin Scaffold in the Continuous Flow.
The continuous flow experimental setup consisted of the capillary in
which the scaffold was immobilized, syringe pump for pumping the
fluid at constant flow rate, a digital manometer (AZ-Instruments),
diffused light for back illumination, and a high resolution camera
Our previous use of glutaraldehyde-cross-linked ferritin in
this procedure gave small (∼several hundred micrometers in
size), brittle, and unstable structures in water. We report here a
significantly improved protocol that allows for the first time the
synthesis of large self-standing macroporous monolith gel from
ferritin. These three-dimensionally connected strands of ferritin
are robust and demonstrate enhanced thermal stability and
tolerance of organic solvents. Organization of ferritin into
scaffolds dramatically simplifies the purification protocols
associated with replacement of the iron oxide nanoparticle
inclusion by catalytically active zerovalent transition metal
nanoparticles. Further, we demonstrate heterogeneously
catalyzed synthesis of organic compounds using monoliths of
Fe O - or Pd-loaded ferritin. The scaffold macropores minimize
(Sony α-37 with DT 18−55 mm lens). The inlet of the capillary had a
Y-shape such that one segment was connected to the pump while the
second segment was attached to an inline digital manometer. The
pressure drop across the capillary was measured using a digital
manometer and the reaction progress was monitored by visually
tracking the color change. The capillary was held vertical and the flow
was downward to facilitate sample collection.
2
3
pressure drop across the monolith while facilitating substrate
and reagent transport. Finally, we demonstrate that the
immobilization of Pd@apo-ferritin scaffolds inside a continuous
flow hydrogenation reactor allows fast hydrogenation of alkenes
in quantitative yields.
Samples were imaged using a Quanta 200 3D scanning electron
microscope. TEM measurements were done at 100 kV on an FEI
Technai F30. An LSM 710 Carl Zeiss laser scanning confocal
microscope (LSCM) was used to image the fluorescent samples. FT-
IR spectra were recorded on a Perkin-Elmer FT-IR spectrum GX
instrument by making KBr pellets. Pellets were prepared by mixing 3
mg of sample with 97 mg of KBr.
Gas chromatography were performed on an Agilent 7890
instrument equipped with a hydrogen flame ionization detector and
HP-5 capillary column (30m × 0.32 mm × 0.25 μm, J & W Scientific)
and nitrogen was used as carrier gas at a flow rate of 1 mL min . The
injector and detector temperature were maintained at 250 °C and
operated in splitless mode. GC-MS were performed on Agilent 5975C
mass selective detector interfaced with Agilent 7890A GC in similar
conditions using a HP-5-Ms capillary column (30 m × 0.32 mm × 0.25
μm, J & W Scientific). Reaction mixtures were quantified from a
standard calibration curve that was plot using pure reactants and
products.
ICP experiments were performed on a Thermo IRIS Intrepid
spectrum apparatus. The typical procedure used was freeze-drying of
sample (3 mg) followed by dissolving in 1 mL of aqua regia. The
resultant solution was diluted to 50 mL, and the quantitative analysis
of Fe and Pd in scaffold was performed using ICP.
EXPERIMENTAL SECTION
■
Materials and Methods. General Procedure for Synthesis of
Macroporous Hydrogel Using Ferritin Based Nanoparticles.
Typically, a 10% (by weight) 55 mg of aqueous dispersion of ferritin
was added to surfactant C E (45 mg, calculated such that the
−1
12 9
surfactant to water ratio was 1:1) and homogenized in a water bath at
0 °C. To this poly(ethylene glycol) diglycidyl ether (4 mg) was
5
added and the sample was then cooled to room temperature in a
feedback controlled convective oven. The particle concentration in the
overall composite is ∼10 wt %. The sample was then allowed to cross-
link on standing at room temperature for 24 h. Finally, the scaffold was
washed repeatedly with water (6−7 times) to remove the surfactant
and afford the free-standing three-dimensional macroporous material.
Synthesis of Apo-ferritin Scaffold from Ferritin Scaffold. An apo-
ferritin scaffold was prepared by demineralization of ferritin scaffold by
23
following the reported procedure. Ferritin scaffold was placed in 200
mL of sodium acetate buffer 0.1M, pH 4.5 and purged with nitrogen
for 30 min. Then 100 μL of thioglycolic acid (TGA) was added while
3
Continuous flow hydrogenation was carried out using H system of
continuously purging with N . After 2 h, further 100 μL of TGA was
Thales Nano Ltd., where the catalyst (60 mg of Pd@apo-ferritin) was
2
added, and the N purge was continued for another hour. Finally, fresh
immobilized inside a stainless steel cylindrical cartridge. In a typical
2
4
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Figure 1. Synthesis of porous ferritin scaffold proceeds via ferritin cationisation using N, N′- dimethyl-1, 3-propanediamine followed by dynamic
templating to assemble the ferritin nanoparticles into a three-dimensional macroporous scaffold. (A) Photograph of ferritin scaffold. (B) SEM image
of cross-linked self-standing scaffold after template removal. (C) Confocal micrograph image of self-standing apo-ferritin scaffold. (D) TEM image of
a section of scaffold (phosphotungstic acid stained).
hydrogenation reaction, a continuous-flow of alkene (80 mg in 50 mL
methanol) was combined with hydrogen (generated in situ from the
electrolysis of water) and was passed through the cartridge packed
with Pd@apo-ferritin at 30 °C and 29 bar pressure. The flow rate of
the alkenes was maintained at 0.2 mL/min and the product was
collected continuously into a collection tube. The methanol was
secondary amino groups by carbodiimide-mediated conjugation
with N,N′- dimethyl-1,3-propanediamine (DMPA, SI-1B).
24
Using this methodology, about 240 secondary amine groups
were installed on the surface of the ferritin nanoparticle. The
resulting amine functionalized particles (8 wt % aqueous
dispersion) was assembled into a macroporous scaffold using
dynamic templating by ring-opening of the terminal epoxide
groups of a poly(ethylene glycol) diglycidyl ether cross-linker
1
subsequently removed, and the product was quantified using H NMR.
Rheological measurements were made using the ARES, a rotary
parallel plate controlled strain rheometer from TA Instruments.
Ferritin gel samples were prepared as 8 mm disks and were loaded on
the rheometer for oscillatory measurements. Measurements were done
on wet hydrogels at room temperature (25 °C) and care was taken to
minimize the evaporation of water during the experiment. At first, a
strain sweep was performed by increasing the strain from 0.5 to 100%
22
(Figure 1), as reported earlier.
Removal of the surfactant by extensive water washing gave
self-standing 3-D macroporous ferritin scaffolds as soft gels,
approximately 1 cm long (Figure 1A). No sign of gelation was
observed in analogous reactions in which surfactant was
omitted (SI-1B), suggesting that protein localization at the
−1
at a frequency of 10 rad s . We then performed a frequency sweep
−1
from 1 to 100 rad s at a strain amplitude of 0.75%.
H domain boundaries is critical for efficient cross-linking and
Small angle X-ray scattering was performed on a BrukerNanostar,
equipped with a rotating anode (Cu Kα radiation), three pinhole
collimation and a 2D multiwire (HiStar) detector (calibrated using
silver behenate standards). Data was collected from the wet ferritin
hydrogel loaded in a thin (10 μm) walled quartz capillary. The data
was circularly averaged, and the 1D scattered intensity is presented
after background subtraction, corrected for sample transmission.
Small-angle X-ray scattering (SAXS) data was analyzed using SASFIT.
1
formation of macroporous materials.
The ferritin-based gel material was characterized in several
ways. To study the bulk three-dimensional structure of the
ferritin network, the scaffold was made with apo-ferritin and
examined by confocal microscopy using the protein’s natural
fluorescence. The resulting images showed the apo-ferritin
scaffold as thick strands organized into networks with pore
dimensions ≈55 μm (Figure 1C). Scanning electron micros-
copy (SEM) obtained after freeze-drying the ferritin scaffold
revealed a network structure comprised of a dense mesh of
strands, with an average strand thickness of about 4.0 μm
(Figure 1B). Image analysis of multiple representative SEM
micrographs showed an average macropore diameter of ≈55
μm (Figure 1B; Figure SI 2). The organization of the ferritin in
the scaffold was investigated using small-angle X-ray scattering
(SAXS) (Figure 2B). SAXS from a dilute dispersion of ferritin
is shown next to that from the scaffold, for ease of comparison
(Figure 2b). The scattered intensity from the ferritin dispersion
was fitted to a model for monodisperse core shell particles with
a core radius of 2.6 nm and a shell thickness of 2.8 nm. Details
RESULTS AND DISCUSSION
■
Synthesis and Characterization of Large Centimeter-
Sized Macroporous Ferritin Scaffold. Stable chemical
linkages and high cross-linking density are required to make
large scaffolds with the mechanical properties required for solid
biocatalysts. Because the ferritin surface displays relatively few
amine groups (the pI of the particle is 4.5), we hypothesized
that the limited mechanical and aqueous stability of ferritin
scaffolds prepared using glutaraldehyde followed by NaCNBH3
treatment was due to low cross-linking density between
nanoparticles. We therefore converted the carboxylate groups
of aspartic and glutamic acid residues on horse spleen ferritin to
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retention of ferritin’s characteristic α-helical secondary
1
6,18
structure
(Figure SI 1).
Dynamic rheological tests indicate the gel-like nature of the
ferritin hydrogels. From frequency sweep measurements to
characterize the mechanical response of the gel, we observe that
the solid modulus (G′) exceeds the loss modulus (G″) by
about an order of magnitude over the entire experimental
−
1
frequency range 1−100 rad s (Figure 2A, Figure SI 4). This
indicates a largely elastic response to small deformations,
characteristic of the network structure of a gel. The solid
0
.08
modulus shows a weak frequency dependence (G′ ∼ ω ), and
tan δ = G″/G′ is approximately frequency independent, as
expected for a gel. The solid modulus is around 3000 Pa,
indicating that the ferritin network forms a soft hydrogel.
Synthesis of Pd Nanoparticle Embedded Apo-ferritin
Scaffold. Apo-ferritin is known to function in solution as a
nanoreactor for the synthesis of encapsulated monodisperse
7
transition metal nanoparticles such as Pd and Au in its core. A
variation of the published metalation procedure was used to
install Pd nanoparticles in the macroscopic apo-ferritin gel.
Instead of making the gel from the apoprotein, the regular
ferritin monoliths were demineralized by incubation with
23
thioglycolic acid. The 3D structure of the resulting colorless
material was unchanged by SEM and confocal microscopy;
EDX analysis showed complete removal of Fe (Figure SI 7;
2
+
Figure SI 9). Incubation of the apo-ferritin scaffold with Pd
salt followed by reduction with NaBH solution led to the
4
0
formation of encapsulated Pd in the ferritin cages comprising
the gel material, indicated by a change in color of the entire
scaffold to black. Extensive washing with water and organic
solvents was performed to remove metallic Pd formed by
uncontrolled reduction on the outer surface of the particles.
Elemental analysis (ICP) showed 0.12 mg Pd per mg of scaffold
before washing and 0.047 mg per mg of scaffold after washing.
TEM of this purified Pd@apo-ferritin composite material
showed presence of relatively monodisperse Pd nanoparticles of
Figure 2. Rheology study was done using ferritin scaffold in water. (A)
Frequency sweep of Ferritin @0.75% strain. (B) SAX scattering curves
for native ferritin 5 wt %, ferritin scaffold and apparent structure factor.
At low q large increase in the scattering indicates that ferritin
nanoparticles are aggregating.
2
−3 nm diameter inside the apo-ferritin corona (Figure SI 8).
The ease and reproducibility of this procedure stands in marked
contrast to the preparation of Pd-filled ferritin proteins in
7
of the fit are provided in the Supporting Information (SI)
solution. In those cases, purification after Pd encapsulation is
(
Figure SI 5). Scattering from the ferritin scaffolds showed a
laborious and requires several chromatographic purification
steps to separate the desired Pd@apo-ferritin from Pd
nanoparticles formed in solution.
−1
peak around 0.07 A , that arises from the aggregation of
individual ferritin units in the network (Figure 2b). We
obtained an apparent structure factor, by dividing the scattering
from the scaffold by the scattering from the dilute ferritin
dispersion. This apparent structure factor shows a power law
decay at low q, with S (q) ∼ q , suggesting a surface fractal
structure for the “walls” of the ferritin network. The correlation
Catalytic Activity of Ferritin and Pd@apo-ferritin
Scaffold for Oxidation and Suzuki−Miyaura Cross
Coupling Reaction. In order to expand the applicability of
ferritin-encapsulated metal catalysts, we evaluated the stability
of this scaffold toward various solvents and elevated temper-
atures. The macroporous ferritin scaffold was found to be stable
toward overnight storage at room temperature in acetonitrile,
ethanol, and toluene, and toward incubation in acetonitrile at
80 °C for 24 h (Figure SI 1). This encouraged us to test these
scaffolds as heterogeneous catalysts in oxidation and C−C
bond formation reactions previously found to be performed by
−3.3
A
peak is modeled using an interference function similar to
25
previous literature reports, which gives an interparticle
distance of 7.1 nm (Figure SI 6). This value is smaller than
the ferritin size obtained from the dilute dispersion SAXS (10.3
nm), in accordance with a previous report on metal mediated
assemblies of cage-like proteins. Transmission electron
microscopy (TEM) showed the scaffold walls to be composed
of ferritin assemblies, with the protein corona encapsulating
dark 4−5 nm iron oxide cores (Figure 1D). Energy-dispersive
X-ray spectroscopy (EDX) analysis of the ferritin scaffold
showed the presence of Fe and P from the iron oxide/
phosphate cores of the protein as well as S from the protein
2
6
27
iron oxide nanoparticles. Most of these reactions have been
limited to the use of water-soluble substrates since native
ferritin protein denatures and precipitates in pure organic
solvents. Also, examples of surface modified ferritin which
displays stability in nonpolar organic solvent are rare and their
28
catalytic activity has not been studied. The organic stability of
the cross-linked materials allowed us to test the oxidation of the
hydrophobic substrate 2,3,6-trimethyl phenol (TMP, only
sparingly soluble in water at neutral pH) to its corresponding
(
Figure SI 3). The intact nature of the protein shells was
−1
suggested by FTIR, which showed peaks at 1650 cm and
550 cm− (amide I and amide II bands) consistent with the
1
1
4
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Figure 3. Synthesis of Pd@apo-ferritin scaffold via demineralization of ferritin scaffold followed by encapsulation of Pd nanoparticles inside the core
of apo-ferritin. The scaffolds were used for the oxidation of 1(a) 2,3,6-trimethylphenol and for the Suzuki−Miyaura cross-coupling reaction. 2a (R =
NH , R′ = H, X = I), 3a (R = OCH , R′ = H, X = Br, 4a (R = OCH , R′ = OCH , X = Br).
2
3
3
3
quinone, trimethyl-1,4-benzoquinone (TMQ), a key inter-
mediate for the industrial production of vitamin E (Figure 3).
TMP (0.05 mmol) and H O (3 equiv) were added to the
more cycles, with no change in product yield (Table 1). In
contrast, the attempted oxidation of TMP to TMQ using the
native ferritin protein and H O (in water at 80 °C), gave rise
2
2
2
2
ferritin scaffold (1 mg; 2.5 μmol Fe) in acetonitrile and the
reaction mixture was heated at 80 °C. The formation of
quinone was indicated by a change in solution color to dark
yellow. GC and GC-MS (Figure SI 10) analysis of the reaction
mixture showed that approximately 80% of the starting TMP
was consumed, approximately 72% of which was converted to
the desired product TMQ (Table 1). No TMQ was observed in
to <10% of the expected amount of TMQ. GC-MS analysis
revealed the formation of 2,2′,3,3′,5,5′-hexamethyl-[1,1′-
biphenyl]-4,4′-diol as a side product, indicative of radical
based C−C coupling (Figure SI 10). Furthermore, ferritin
protein precipitated during the course of the reaction,
indicating denaturation under the reaction conditions.
Taken together, these data suggest that the cross-linked
macroporous ferritin scaffold is a true reusable heterogeneous
catalyst for the synthesis of TMQ with several advantages. It
allows the reaction to be carried out in acetonitrile and at a high
temperature while retaining the structural integrity of the
scaffold.
Table 1. Oxidation and Suzuki−Miyaura cross Coupling
a
Reaction with Ferritin and Pd@apo-ferritin
b
entry
reactant
product
cycle
conversion (%)
yield (%)
1
2
3
4
5
6
7
8
1a
1a
1a
2a
2a
2a
3a
4a
1b
1b
1b
2b
2b
2b
3b
4b
1st
79
79
75
78
74
72
75
71
72
70
70
87
83
83
81
80
Similarly, the Pd@apo-ferritin scaffold was evaluated in the
Suzuki−Miyaura cross-coupling reaction of p-substituted
iodoaniline and phenylboronic acid in water (Figure 3), a
2nd
3rd
1st
7
process known to be catalyzed by other Pd nanoparticles.
2nd
3rd
1st
Incubation of iodoaniline (2a) with phenylboronic acid in
water containing the Pd@apo-ferritin scaffold (1.17 μmol Pd,
0
.007 mol % of Pd nanoparticle relative to (2a) at 75 °C
1st
overnight gave rise to the product amino-biphenyl (2b) in 87%
yield. As in the case of the native ferritin scaffolds, neither
leaching of Pd nor a change in the secondary structure of the
ferritin coat protein was observed, and at least two addition
cycles of catalysis could be performed without change (Table
1). As expected, TEM of the scaffold after the reaction showed
no agglomeration of the Pd nanoparticles (Figure SI 11), which
would otherwise occur if they were not shielded by the cage
protein. The Suzuki−Miyaura cross-coupling reaction was also
extended to the substituted anilines 3a and 4a with similar
product yields (Figure 3).
a
For entry 1−3 Ferritin scaffold was used while for entry 4−8 Pd@
b
apo-ferritin scaffold was used. Yield was calculated based on %
conversion.
control reactions omitting either H O or the ferritin scaffold.
2
2
ICP analysis revealed no iron in solution, suggesting that the
reaction was catalyzed by the iron oxide nanoparticles present
in the scaffold and not by iron salts leached out during the
reaction. FT-IR analysis showed the same spectra before and
after completion of reaction, showing no gross change in
protein secondary structure during the first cycle. The
recovered ferritin scaffold was then used as a catalyst for two
Macroporous Scaffold for Continuous Flow Synthesis.
In the pharmaceutical and fine chemicals industries, process
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Chemistry of Materials
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−
1
intensification through conversion of traditional batch
processes to continuous flow operation is receiving significant
(flow rate 0.2 mL min ), representing approximately 12195
turnovers of substrate per Pd nanoparticles (Figure SI 1B). For
cinnamyl alcohol (Table 1, entry 2), the yield of the reaction
remained unchanged (99%) for an additional 15 reaction
cycles.
2
9
attention. As in many flow processes, we anticipated that our
ferritin-based gels would allow enhanced heat and mass
transfer, due to the hydrodynamics of flow through the random
pore structure without significant back pressure restrictions, a
consequence of the high porosity of the gel microstructure. To
test this, we performed oxidation of colorless TMB to its one-
Continuous flow transfer hydrogenation with palladium
nanoparticle loaded microreactor monolithic glass/polymer
31
composite has been demonstrated previously. However, upon
progression of the reaction the Pd nanoparticles leached out
and the rate of leaching was dependent on the polarity of the
solvent and acidity. In our scaffold, we have demonstrated that
Pd nanoparticles does not leach out under demanding reaction
conditions. Hence, we were able to perform 15 catalytic cycles
of hydrogenation with our scaffold without any loss of
reactivity.
30
electron oxidized blue product in the presence of H O , using
2
2
the ferritin scaffold immobilized inside a glass capillary, as
shown in Figure SI 12.
The reactant was passed through the capillary at different
flow rates using a syringe pump. The pressure drop across the
capillary was a modest ≈0.0039 atm for a monolith of 1 cm
length. The reaction progress was monitored by visually
tracking the color change. For the scaffold having >90% porous
flow volume, the reaction was seen to proceed even for a
residence time of 5.5 s. We were able to carry out these
transformations for several hours in continuous flow mode
without change in catalyst activity or scaffold structure.
Having established that our ferritin gel monoliths can
combine the advantages of continuous flow operation with
low pressure drops, we employed Pd@apo-ferritin gels for an
industrially important organic transformation, the continuous
CONCLUSION
■
In summary, we demonstrate here the first synthesis of large
(centimeter-sized) self-standing macroporous scaffold mono-
liths from ferritin bionanoparticles. The monolith allows easy
replacement of iron oxide nanoparticles with other catalytically
active late transition metal nanoparticles such as Pd, facilitating
the catalysis of several organic transformations both in batch
and continuous mode. This demonstration illustrates several
practical advantages to solution-phase nanoparticle-based
catalysis, including (i) synthesis and metal-swapping procedures
performed without the need for tedious chromatographic
purification; (ii) high stability of encapsulated metal nano-
particles against leaching under the reaction conditions,
enabling to use in continuous flow configuration. The
methodology for monolith preparation is general and should
allow the synthesis of macroporous scaffolds from polymer
coated metal nanoparticles as well, although it will be
interesting to see what differences may emerge when the
component particles are not as monodisperse as the protein
nanocages used here.
3
flow hydrogenation of alkenes (Figure 4). Using the H system
(
Thales Nano Ltd.) at 30 °C and 29 bar pressure (Figure SI
1
1
3), three substrates were hydrogenated as shown in SI Table
. The use of 60 mg of monolith allowed the quantitative
hydrogenation of 80 mg of each substrate in 50 mL methanol
ASSOCIATED CONTENT
■
*
S
Supporting Information
Ferritin modification, FT-IR spectra, EDX, SEM, and TEM
image with pore and nanoparticles size distribution, fluo-
rescence spectra, SAXS fitting, GC-MS data and set up for
continuous flow. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
E-mail: ss.sengupta@ncl.res.in.
E-mail: g.kumaraswamy@ncl.res.in.
*
*
Author Contributions
The manuscript was written through contributions of all
authors.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Authors thank Prof M. G. Finn for discussion and help with
writing the manuscript. They also thank Chandrababu Naidu
and Dr. C. V. Ramana for help regarding continuous flow
hydrogenation reaction. S.S.G. thanks Indo-Korean project
INT-Korea-P-15 from DST, New Delhi, for funding. A.A.K.
Figure 4. Schematic representation for the continuous flow hydro-
genation reaction of different substrates with immobilized Pd@
3
apoferritin scaffold using H system (Thales Nano Ltd.).
4
818
dx.doi.org/10.1021/cm403240j | Chem. Mater. 2013, 25, 4813−4819

Chemistry of Materials
Article
thanks network project CSC0123 for funding. S.K. thanks
CSIR, New Delhi, for fellowship.
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