Magnetic MOF microreactors for recyclable size-selective biocatalysis

Article abstract of DOI:10.1039/c4sc03367a

In this contribution we report a synthetic strategy for the encapsulation of functional biomolecules within MOF-based microcapsules. We employ an agarose hydrogel droplet Pickering-stabilised by UiO-66 and magnetite nanoparticles as a template around which to deposit a hierarchically structured ZIF-8 shell. The resulting microcapsules are robust, highly microporous and readily attracted to a magnet, where the hydrogel core provides a facile means to encapsulate enzymes for recyclable size-selective biocatalysis.

Full text of DOI:10.1039/c4sc03367a

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Magnetic MOF microreactors for recyclable size-
selective biocatalysis
Cite this: DOI: 10.1039/x0xx00000x
a†
a
a
a*
Jia Huo, Jordi Aguilera-Sigalat , Samir El-Hankari and Darren Bradshaw
In this contribution we report a synthetic strategy for the encapsulation of functional
biomolecules within MOF-based microcapsules. We employ an agarose hydrogel droplet
Pickering-stabilised by UiO-66 and magnetite nanoparticles as a template around which to
deposit a hierarchically structured ZIF-8 shell. The resulting microcapsules are robust, highly
microporous and readily attracted to a magnet, where the hydrogel core provides a facile
means to encapsulate enzymes for recyclable size-selective biocatalysis.
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
adsorption directly into MOF pores necessarily restricts the size
Introduction
7
of the proteins that can enter the pores which may need to
1
0
undergo conformational changes that could compromise
biomolecule activity. An alternative strategy to overcome these
challenges is to configure MOFs into hollow microcapsular
structures, where encapsulation of biomolecules within the
capsule interior will not significantly impact upon the inherent
porosity of the MOF shell and permit larger proteins to be used.
Biocatalysis with natural enzymes is extremely attractive for
sustainable chemistry due to high selectivity and reactivity
under mild reaction conditions, minimizing waste and energy
1
input.
However, these highly functional biological
macromolecules do not always operate optimally in the organic
solvents typically employed in synthetic chemistry and their
2
recovery can be problematic. Immobilization or encapsulation
MOF capsules have previously been prepared around sacrificial
of enzymes for biocatalysis is a proven strategy to overcome
these limitations, and although a diverse range of materials
1
1
polystyrene cores , by interfacial synthesis in microfluidic
1
2
13
3
devices , one-pot assembly around emulsion droplets and by
have been employed to enhance enzyme activity and stability ,
1
4
spray-drying. None of these techniques however is amenable
to the encapsulation of biomolecules: for example, solid
sacrificial cores cannot readily encapsulate the desired cargo
and the heat required to induce crystallization of a MOF shell
or the presence of surfactant amphiphiles for the templating of
hollow structures, have the potential to destabilize proteins. By
contrast, we recently reported that MOF nanoparticles (NPs)
can stabilize oil-in-water Pickering-type emulsions to form
coordination-based analogues of colloidosomes, which can act
as polymerization microreactors to form robust MOF-polymer
composite capsules suitable for encapsulation and pH-triggered
the need to develop new methods and hybrid capsules with
hierarchically structured shells, ordered porosity and
controllable compositions is important to drive innovation in
this and wider areas of biotechnology.
Metal-organic frameworks (MOFs) are crystalline microporous
networks formed by the assembly of metal ions and organic
linkers, which have high surface area and readily tuneable
4
structures, pore sizes and compositions. While this ensures
their widespread use in applications traditionally associated
with porous materials (e.g. molecular storage, separation,
1
5
5
release of dye molecules. While there are several reports of
Pickering stabilized biocatalysis microreactors using diverse
delivery and catalysis) , it also qualifies them as excellent
potential supports for enzymatic biocatalysis. There remain few
reports of immobilization of active enzymes onto MOF
1
6
17
particles and polymersomes , the exceptional microporosity
and tuneable properties of MOFs provide a unique opportunity
to develop novel potentially multifunctional hybrid composites
with controllable structures, compositions and pore sizes. In
this contribution we report a strategy for the encapsulation of
functional biomolecules within MOF-based microcapsular
structures. By utilizing a Pickering-stabilised hydrogel droplet
we can deposit a hierarchically structured MOF shell to form
robust free-standing capsules with high microporosity that are
easily recovered by a magnet owing to a small amount of
supports, and current strategies involve the adsorption of
6
biomolecules into mesoporous
and ultra-large pore
7
frameworks , covalent surface modification of MOF crystals
with proteins or host-guest interactions between tag-group(s)
8
on the enzyme and MOF pores to anchor the large functional
9
molecules to the surface. These post-synthetic methods are not
without their drawbacks, and typically lead to significant
reductions in porosity as the large protein molecules occupy
pores or block (surface) apertures to the available framework
microporosity; although not necessarily a barrier to function,
this could be an issue for the selectivity and/or molecular
diffusion of composite biocatalytic materials. Further,
1
8
incorporated magnetite.
(Figure 1) The hydrogel core
provides a facile means to encapsulate large biomolecules
under mild conditions that are not accessible to other MOF
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Figure 1. Synthesis and characterization of the magnetic MOF microcapsules. (a) Optical microscopy image of agarose hydrogel droplets Pickering-stabilized by UiO-
6/Fe nanoparticles; (b) idealized representation of the two-step synthesis protocol to form the ZIF-8 shell, (c) SEM image of the intact capsules and (d) the
6
3 4
O
crushed sample, showing their hollow nature and hierarchically structured shell comprising of (e) a network-like interior and (f) a dense outer-layer of ZIF-8; (g) PXRD
data of the microcapsules compared to the single components, revealing the presence of both MOFs; (h) demonstration of the magnetic recovery of the capsules; (i)
Nitrogen adsorption-desorption isotherm of the capsules at 77 K (black squares, adsorption; red squares, desorption). The inset shows the BJH pore size distribution
calculated from the adsorption branch of the isotherm.
1
9
66
[Zr O (OH) (BDC) ,
where
BDC
=
1,4-
6
4
4
6
capsule formation methods. The resulting biomolecule loaded
capsules can be employed for recyclable size-selective
biocatalysis due to the high integrity of the microporous
capsule shells, which regulate molecular access to and from the
capsule lumen. As a direct consequence of the capsule structure
and composition, we also find that enzyme activity is modestly
enhanced compared to the control samples.
benzenedicarboxylate] and Fe O . (Figure 1a) Heptanoic acid
3
4
functionalised UiO-66 (figure S1) was selected for its ready
15
dispersibility in paraffin oil and to provide a surface for
further MOF growth, and Fe O4 to facilitate magnetic
3
separation during capsule preparation and subsequent
18
application. The average diameter of the spherical hydrogel
droplets stabilized by 1 wt% added UiO-66/Fe O NPs (wt/wt
3
4
ratio 9:1) is 27.4 µm; however, this is tuneable by modulating
the quantity of added particles. (Figure S2)
Results and discussion
MOF capsule formation and characterisation
2
0
As shown in figure 1b-f, a shell of ZIF-8 , [Zn(2-MeIm) ]
2
where 2-MeIm = 2-methylimidazole, is deposited around the
For the encapsulation of enzymes within MOF capsules, we
employ an inverse phase Pickering emulsion containing 1.5
wt% agarose hydrogel droplets in a continuous paraffin oil
phase which are stabilized by pre-hydrophobized NPs of UiO-
UiO-66/Fe O4 Pickering-stabilized hydrogel core. For ZIF-8
3
shell formation, we utilize a two-step synthesis protocol (figure
1b) that effectively separates out the framework nucleation and
growth steps under solvent control at low temperature (-20°C).
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The low reaction temperature improves overall shell integrity, To evaluate capsule shell integrity and hence their potential in
maintains gelation of the core during shell formation and size-selective applications, we used large dye molecules as
prevents deactivation of any incorporated biomolecules. The fluorescent probes of molecular uptake and release. (Figure 2)
i
first step is conducted in isopropanol ( PrOH), and even at - Water soluble Rhodamine B (RhB) was loaded into the
2
0°C leads to the rapid precipitation of ZIF-8 (figure S3) which capsules by inclusion within the UiO-66/Fe O4 stabilized
3
immediately provides additional stabilization to the UiO- hydrogel. Despite a rather low 0.58% encapsulation efficiency
6/Fe O Pickering emulsion as ZIF-8 particles quickly of RhB as a result of significant leaching from the hydrogel due
6
3
4
aggregate around the hydrogel cores. This is followed by a to high solubility in the alcohols used for ZIF-8 shell formation,
slower growth-dominated step in 2-butanol (2-BuOH) (figure this remains sufficient to permit localization by confocal laser
S3) to yield highly robust free-standing capsules. Scanning scanning microscopy (CLSM) and determine any leaching
electron microscopy (SEM) images reveal intact spherical through the shell. The CLSM images in figure 2a reveal that the
structures of average size 37.6 ± 8.6 µm, which are RhB is concentrated around and/or within the interior of the
demonstrably hollow when crushed (figure 1c,d and S4 - S6). capsule shell as evidenced by the observed red halo and
The shell is 4 – 5 µm thick (by SEM) and has a clear bimodal fluorescence intensity profile, consistent with a strong
2
3
hierarchical structure, reminiscent of supported MOF interaction between the shell and the dye.
Control
2
1
membranes. The interior of the shell is composed of a experiments with Fe O4 only and UiO-66 only Pickering-
3
continuous mesoscale network of aggregated ZIF-8 NPs < 100 stabilized hydrogels reveal a similar fluorescence profile for
nm in size (figure 1e and S5, S6), consistent with the observed UiO-66, whereas
a homogeneous distribution of RhB
very rapid nucleation of the framework. Overlaying this throughout the capsule lumen is observed when only Fe O NPs
3
4
network-like interior is a thin (~ 400 nm) but very dense are used. (Figure S11) This confirms the role of the MOF
coating of much larger and highly-intergrown ZIF-8 crystals particles in dye accumulation in the capsule shell, and since
(
figure 1f), resulting from much slower framework growth in 2- RhB is too large to enter the micropores of either ZIF-8 or UiO-
BuOH. This dense over-layer gives the capsules additional 66, this is most likely to be surface adsorption arising from
structural stability, and strongly suggests these could find utility interactions between the abundant organic functionalities of the
in size-selective applications where molecular access is hybrid framework particles and the aromatic dye.
controlled by the highly regular micropores of the ZIF-8
sodalite topology as previously reported for catalysis by ZIF-8
2
2
encapsulated noble metal NPs.
The capsules were further characterized by powder X-ray
diffraction (PXRD), indicating the presence of Bragg
reflections corresponding to the cubic unit cells of both the
deposited ZIF-8 shell and the UiO-66 NPs employed for
emulsion stabilization (figure 1g), thus confirming they have a
dual-MOF composition; this was also demonstrated by FTIR
(
figure S7). A broad Bragg reflex of relatively low intensity
observed at 2Θ ~ 36° is assigned to the minor Fe O4
3
component, which gives the capsules their slight brown/grey
coloration and permits their ready attraction to a permanent
magnet (figure 1h). The capsules have an overall composition
of 1:32:63 for Fe O :UiO-66:ZIF-8 by weight as determined by
3
4
ICP. Both MOFs are incorporated into the composite, and SEM
images clearly reveal the presence of aggregated UiO-66 NPs
clustered around the interior of the capsule shell. (Figure1b, S6)
Thermogravimetric analysis (TGA) indicates the capsules
largely decompose in a single step in the temperature range 320
–
6
7
500°C, and a small step is also observed between 500 –
50°C. (figure S8) Nitrogen adsorption isotherms (figure 1i) at Figure 2. Encapsulation properties of the MOF composite microcapsules. (a)
CLSM image of capsules loaded with RhB, and inset a typical fluorescence profile;
(b) RhB retention vs. capsule incubation time in water at ambient temperature;
(c) CLSM image and fluorescence intensity profile of the highlighted capsule
7K demonstrate the capsules are highly microporous with an
2
-1
apparent BET surface area of 1452 m g and a pore volume of
3
-1
0.82 cm g . A slight hysteresis is observed upon desorption,
(
inset) after soaking capsules in methanolic solutions of FITC; (d) CLSM image of
and application of a Barrett-Joyner-Halenda (BJH) pore size
distribution analysis (figure 1i, inset) reveals pores across the
meso- and macroporous regimes consistent with the
hierarchical structure of the capsule shell observed by SEM
the capsules loaded with FITC-labelled CalB, and inset the fluorescence profile of
the highlighted capsule.
(
figure 1d-f). The capsules are highly stable, retaining their Following acid dissolution of the capsules, the RhB loading
-1
spherical shape upon solvent removal or exchange (figure S9), level was determined as 0.014 µmolg by UV/vis spectroscopy,
and the MOF shell does not degrade after soaking in organic which did not change after 24 hrs soaking in water (figure 2b)
solvents, water or phosphate-buffered saline (PBS) (figure S10) confirming the intact nature of the capsule shell. The
consistent with its composition.
microcapsules were also immersed in dye solutions to
investigate diffusion into the interior from bulk solution. CLSM
images show the interior and shell of the capsules remain
totally dark when soaked in methanolic solutions of fluorescein
Capsule loading and shell integrity
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isothiocyanate (FITC) (figure 2c) or aqueous RhB (figure S12). encapsulation process is expected to lead to increased enzyme
By contrast capsules isolated after step one of the synthesis in loading.
i
PrOH only, show significant FITC uptake into the meso-
/
macroporous network-like shell structure (figure S13)
The biocatalytic performance of CalB@cap and the size
selectivity of the microcapsule shell were assessed by carrying
out transesterification reactions between a pair of small
substrates and a pair of larger ones as shown in figure 3a and d,
respectively, and figure S17. The following experiments were
verifying the importance of the intact microporous ZIF-8 outer
layer to control molecular diffusion into and out of the
capsules.
The UiO-66/Fe O stabilized hydrogel core provides a facile also performed: CalB in agarose gel droplets Pickering-
3
4
means for the encapsulation of diverse and functional stabilized by UiO-66/ Fe O (CalB@Pick) and CalB in agarose
3
4
biomolecules into the robust MOF microcapsules. We only (CalB@gel) as positive controls and empty MOF
investigated this by adding green fluorescent protein (GFP) and microcapsules (cap) as a negative control. (Figure S18)
the FITC tagged enzymes Candida Antarctica lipase B (CalB) Biocatalysis reactions were carried out at r.t. in acetone and
1
and β-galactosidase (β-gal) to the Pickering-stabilized agarose progress monitored by H NMR; products were further
hydrogels prior to shell formation. CLSM images of the loaded identified with ESI-MS. For reaction of
capsules (figure 2d and S14) clearly demonstrate successful positive controls reach 100% conversion to
1
and
2
3
(fig 3a), the
after 12 hrs
encapsulation of the fluorescent proteins and reveal their whereas CalB@cap takes ~ 48 hrs. (Figure 3b and S19-S23)
accumulation around and/or within the interior of the capsule
shells in a similar manner to the dyes. This likely arises from
the composition of the MOF shell, which appears to display an
appropriate hydrophilic/phobic balance to enhance interactions
with encapsulated biomolecules. To investigate whether the
protein molecules were immobilized within interstices between
the aggregated NPs which comprise the interior of the
hierarchically structured shell, CalB loaded capsules were
incubated in more polar MeOH at r.t. overnight. CLSM reveals
the fluorescent enzyme becomes homogeneously distributed
throughout the lumen (figure S15), indicating permeability of
the shell to small molecules and solvent dependent enzyme
mobility within the capsules.
The proteins employed in this study range in molecular weight
from 27 kDa for GFP to 465 kDa for the large homo-tetrameric
enzyme, β-gal. While some reports of protein incorporation into
6
,7
MOFs do exist , even the current largest pore frameworks (viz.
IRMOF-74-XI which has 10 nm pores) will be unable to
accommodate such large molecules as β-gal which has
dimensions of 3.0 x 13.5 x 17.5 nm; hence the configuration of
2
4
MOFs into capsules or other appropriate superstructures
under mild conditions as demonstrated here are clearly
necessary to meet these demands.
Figure 3. Biocatalysis using CalB loaded MOF microcapsules. (a) Reaction scheme
for the transesterification between 1-butanol (1) and vinyl acetate (2) to yield
butyl acetate (3); (b) Observed time dependent conversion of 1 and 2 catalyzed
Biocatalysis
-
1
by CalB under different conditions; (c) Normalized activities (U = µmol·min ) of
the capsules and controls for the formation of 3; (d) Scheme of
transesterification between 3-(4-hydroxyphenyl) propan-1-ol (4) and vinyl
laurate (5) to yield 3-(4-hydroxyphenyl)propyl dodecanoate (6). (e) Observed
time dependent conversion of 4 and 5 catalyzed by CalB under different
CalB is a robust broad-substrate lipase (MW = 33 kDa) widely
employed for the synthesis of organic compounds through
esterification and transesterification reactions in organic
2
5
solvents , making it an excellent model candidate for conditions; (f) Normalized activities of the capsules and controls for the
microencapsulation and biocatalytic studies. (Figure 3) CalB formation of 6.
loaded microcapsules (CalB@cap) were prepared as described,
and were found to maintain a very high degree of porosity
2
-1
This indicates enzyme activity is maintained following ZIF-8
shell formation and suggests that can readily diffuse
through the framework micropores of the intact outer shell.
Magnetic removal of the capsules from the reaction after 10 hrs
(
BET = 1438 m ·g ) (figure S16) unlike previous studies where
1
– 3
enzymes have been physically adsorbed into MOF
6
,26
mesopores
or covalently immobilized on their crystal
8
surfaces , strongly indicating that encapsulation is a superior
strategy to prepare highly porous MOF-enzyme composite
materials. Following capsule degradation, a Bradford assay
revealed a CalB loading of 7 mg·g (0.2 µmol·g ), comparable
to the level observed for enzyme immobilization on some
reveals no further conversion to 3, demonstrating CalB is not
leached from the microcapsules which remain fully intact and
impermeable to FITC (figure S24) following catalysis. As
anticipated, the empty microcapsules without CalB are inactive
for transesterification. The capsules have a CalB loading
efficiency of 30% (Bradford assay) resulting from incomplete
shell formation during synthesis, and while the reduced enzyme
contents largely account for the shallower conversion-time
-
1
-1
2
7
mesoporous silicas but lower than reported for direct
adsorption into MOF mesopores over a two day period. The
6
maximum loading level of CalB into the capsules has not been
fully explored, but inclusion of a higher concentration into the
Pickering-stabilized hydrogel and further optimisation of the
4
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profile observed for CalB@cap, a contribution due to diffusion For transesterification between larger substrates
4
and
with CalB@gel and CalB@Pick was
similar to that observed for , reaching 100% after ~ 12 hrs.
Figure 3e and S27-S31) In this case however, very little
conversion was observed with CalB@cap for the first 12 hrs of
reaction, although after this point a slow conversion to was
5 (figure
through the capsule shell will certainly play an important role.
3d), conversion to 6
3
(
Following normalization to the total protein contents (figure
S25), the specific activity (U·mg ) of CalB@cap is 2.6 times
higher than CalB@gel and 1.7 times greater than CalB@Pick.
-
1
6
observed reaching 7.5% after 48 hrs. The observed induction
period suggests a significantly increased diffusion barrier is
provided by the capsule shell and/or potential blockage of pores
(
Figure 3c) This is attributed to differences in interfacial area,
1
7, 28
as commonly observed in Pickering-derived microreactors.
CalB@gel displays the lowest specific activity due to the
biphasic nature of the reaction where the substrate solution sits
above a hydrogel monolith. (Figure S18) Activity is increased
by a factor of 1.5 through Pickering stabilization of the
hydrogel droplets with UiO-66/Fe O NPs, which present a
larger interface for reaction. (Figure S18) The highest activity is
consequently observed for CalB@cap resulting from the
hierarchically structured microcapsule shell, as previously
reported for polydopamine capsules with highly interconnected
porous wall structures. There is also likely to be an enzyme
stabilizing effect by the capsule shell through hydrophobic
interactions, and the potential to accumulate substrate
molecules around CalB via MOF micropores may further
contribute to the observed enhancement of activity. The
or defects by larger molecules such as
for the controls followed the same trend as for
4
–
6
. Specific activity
; however, with
3
larger substrates the activity for CalB@cap is effectively zero
during the early linear part of the conversion-time profile where
3
4
specific activity is typically calculated. (Figure 3f) Given that
3
and are formed by CalB@gel and CalB@Pick with very
6
similar activity, the clear differences observed for CalB@cap
are directly attributed to the variation in molecular size of both
the substrates and products and the ability of the intact
2
9
(
microporous) capsule shell to regulate their access. The
limiting molecular dimensions for the smaller substrates are
.06 and 4.18 Å for and , respectively, and 4.06 Å for
product . (Figure S17) These sizes are only slightly larger than
the aperture size of ZIF-8 (3.4 Å), and given the uptake of 1-
4
1
2
3
transesterification of
1 and 2 was also carried out in hexane,
3
1
butanol (1) has previously been reported for this framework it
where the specific activity of CalB@cap was less than that
observed in acetone revealing that solvent effects remain
important to the encapsulated biocatalysts. Activity of
CalB@cap in hexane was also enhanced by a factor of 5
relative to the free enzyme (figure S26), further confirming the
role of the MOF shell in enzyme stabilization.
seems likely that these molecules could pass through the
micropores of the dense ZIF-8 outer layer to access the capsule
interior. For the larger molecules
4 – 6, the limiting molecular
cross-section is 8.84 Å (figure S17) so these are expected to be
excluded by the micropores of the capsule shell. While two
3
2
recent reports suggest that oversize molecules such as caffeine
3
3
and Rhodamine 6G can be encapsulated by ZIF-8 arising
from framework expansion or dissociative linker exchange
mechanisms, it is most likely that
4 – 6 pass through very small
pin-hole defects within the dense microporous ZIF-8 outer
layer.
Although size-selective biocatalysis has previously been
reported for oxidation reactions catalyzed by myoglobin
immobilized within the mesoporous cages of a hierarchically
2
6
porous MOF , the present work is a clear proof-of-principle
demonstration that encapsulation of biomolecules using
Pickering-stabilised hydrogels is a viable alternative strategy
for the preparation of highly porous and functional MOF-based
biocatalytic systems.
Figure 4. Catalytic recyclability of CalB@cap for the transesterification of
1 and 2
in acetone. The catalyst was removed following 8 hrs of reaction and conversion Conclusions
1
to
3
calculated by H NMR for each cycle.
In summary we have developed a synthetic route to prepare
hierarchically porous MOF-based capsules around Pickering-
The ease of magnetic recovery of CalB@cap and the absence of stabilized hydrogels for the effective encapsulation of
enzyme leaching prompted us to investigate the catalytic functional biomolecules, and demonstrated their practical utility
recyclability of the MOF-based microcapsules. Following in size-selective and recyclable biocatalysis. Processing of
reaction of
washed and used again for further transesterification reactions. manner reported here, appears to be a very effective strategy to
Conversion yield of reveals very good overall recyclability prepare highly porous functional MOF-biomolecule composite
with 80% of activity maintained after six catalytic cycles materials with significant potential in wider biotechnology
figure 4), comparing well to other Pickering-based applications including drug delivery, tandem chemoenzymatic
1 and 2 for 8 hrs, the capsules were recovered, MOFs beyond simply increasing the framework pore size in the
3
(
1
7
microreactors with encapsulated CalB. In the present case, catalysis and in novel bioseparation and biodegradation media.
given the intact nature of the capsules and demonstrable
heterogeneous nature of the catalysis, we attribute the modest
Acknowledgements
decrease in conversion to the cumulative effects of enzyme
We gratefully acknowledge the European Research Council for
3
0
denaturing by the 1-butanol substrate.
funding (ERC-StG-2010-258613-BIOMOF).
Notes and references
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