Highly active enzymes immobilized in large pore colloidal mesoporous silica nanoparticles

Article abstract of DOI:10.1039/c8nj04585b

Various bio-applications of mesoporous materials (e.g., the immobilization of enzymes or the delivery of biomolecules such as siRNA) require large pores for the successful adsorption of the rather large molecules of interest and protecting the fragile cargo from external forces such as degradation. We describe the facile synthesis of functionalized mesoporous silica nanoparticles with large pores (LP-MSNs) providing high loading capacity for the immobilization of two differently-sized enzymes. The synthesis procedure yields homogeneous core-shell particles of about 100 nm in size with large mesopores (about 7 nm in diameter) and an azide-functionality inside the pores. The LP-MSNs were synthesized employing a co-condensation approach with the rather large micellar template cetyltrimethylammonium p-toluenesulfonate (CTATos). Due to the azide functionality, the LP-MSNs are suitable for bio-orthogonal click chemistry reactions within the porous network. Two different acetylene-functionalized enzymes (sp-carbonic anhydrase (CA) and sp-horseradish peroxidase (HRP)) were immobilized in the pores of the obtained LP-MSNs by a copper-catalyzed 1,3-dipolar cycloaddition reaction. The covalent attachment of the enzymes within the mesopores allowed us to investigate the catalytic performance of the enzyme-silica systems. The enzymes are stable after bioconjugation with the silica support and show high catalytic activity over several cycles for the colorimetric reaction of guaiacol (2-methoxyphenol) in case of LP-MSN-HRP and the hydrolysis of 4-nitrophenyl acetate (NPA) by LP-MSN-CA.

Full text of DOI:10.1039/c8nj04585b

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Highly active enzymes immobilized in large pore
colloidal mesoporous silica nanoparticles†
Cite this:DOI: 10.1039/c8nj04585b
Dorothee Goßl,
Martina Lichtnecker, Hanna Engelke and Thomas Bein
Helena Singer, Hsin-Yi Chiu, Alexandra Schmidt,
´
¨
*
Various bio-applications of mesoporous materials (e.g., the immobilization of enzymes or the delivery of
biomolecules such as siRNA) require large pores for the successful adsorption of the rather large
molecules of interest and protecting the fragile cargo from external forces such as degradation. We
describe the facile synthesis of functionalized mesoporous silica nanoparticles with large pores (LP-MSNs)
providing high loading capacity for the immobilization of two differently-sized enzymes. The synthesis
procedure yields homogeneous core–shell particles of about 100 nm in size with large mesopores (about
7
nm in diameter) and an azide-functionality inside the pores. The LP-MSNs were synthesized employing a
co-condensation approach with the rather large micellar template cetyltrimethylammonium p-toluene-
sulfonate (CTATos). Due to the azide functionality, the LP-MSNs are suitable for bio-orthogonal click
chemistry reactions within the porous network. Two different acetylene-functionalized enzymes
(sp-carbonic anhydrase (CA) and sp-horseradish peroxidase (HRP)) were immobilized in the pores of the
Received 9th September 2018,
Accepted 3rd December 2018
obtained LP-MSNs by a copper-catalyzed 1,3-dipolar cycloaddition reaction. The covalent attachment of
the enzymes within the mesopores allowed us to investigate the catalytic performance of the enzyme–
silica systems. The enzymes are stable after bioconjugation with the silica support and show high catalytic
activity over several cycles for the colorimetric reaction of guaiacol (2-methoxyphenol) in case of LP-MSN–
HRP and the hydrolysis of 4-nitrophenyl acetate (NPA) by LP-MSN–CA.
DOI: 10.1039/c8nj04585b
rsc.li/njc
encapsulation of large biomolecules (i.e. enzymes or siRNA) has
Introduction
1
4–17
been addressed.
Recently, the group of Zhang published
Ordered mesoporous silica materials have attracted increasing a synthesis route for wide-pore stellate MSNs via the use of
attention due to the ability to tailor the structural and textural cetyltrimethylammonium p-toluenesulfonate (CTATos) as tem-
1
8
features including particle size, pore size, pore volume and plate in a near-neutral precursor solution. The resulting
1
–6
surface area. With the possibility to introduce various func- stellate particles show a pore size distribution between 10 to
tionalities into the mesoporous framework, these materials 20 nm and a defined particle size of around 100 nm. With those
have turned into interesting candidates for various biotechno- properties they represent an ideal delivery vehicle for the
4,7–10
19
logical applications.
However, such applications often involve cellular transport of large-sized biomolecules.
fairly large molecules of more than 5 nm in size that may not fit
Here, we describe a delayed co-condensation approach to create
into the pores of established silica nanoparticles (ca. 4 nm). spatially segregated core–shell phenyl- and azide-functionalized
Additionally, the active molecules need to be immobilized while large-pore MSNs (LP-MSNs), which serve as a versatile platform for
preserving their function.
the immobilization of enzymes. Due to their large pores, our
Considerable efforts have been made in the past years functionalized LP-MSNs can be loaded with large bioactive mole-
regarding the control of mesostructural diversity, composi- cules and thus are promising candidates for numerous bio-
tional flexibility and morphology during the synthesis of meso- applications, including biocatalysis.
3
,11–13
porous silica nanoparticles (MSNs).
In recent years the
A promising way to introduce enzymes as biocatalysts in
synthesis of well-defined large-pore mesoporous systems with industrial applications is their immobilization onto inorganic
2
,20–22
small particle sizes (in the size-range of 100–200 nm) for the mesoporous supports.
Organo-functionalized nanostruc-
tured mesoporous silica materials have attracted great atten-
tion among the available inorganic host systems as they offer
outstanding properties for the immobilization and encapsula-
Department of Chemistry and Center for NanoScience (CeNS), University of Munich
LMU), Butenandtstrasse 5–13(E), 81377 Munich, Germany. E-mail: bein@lmu.de
Electronic supplementary information (ESI) available: Fig. S1–S11 (additional
characterization) and calculation A1. See DOI: 10.1039/c8nj04585b
(
2
,23,24
tion of biomolecules.
As the pores of LP-MSNs match the
†
size of enzymes, such silica materials are excellent candidates
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5
for enzyme immobilization, yet their potential in this field and remained highly active after covalent immobilization in
still needs to be explored. Previous studies on established the LP-MSNs.
mesoporous silica materials (e.g. SBA-15) revealed that enzyme
In this work we combine a simple support synthesis with a
confinement within such hosts can increase the stability and very efficient immobilization technique to obtain an active,
reduce denaturation by protein unfolding, one major issue in stable and reusable enzyme–silica system.
2
6
industrial applications of biocatalysts. However, as a con-
sequence of immobilization onto the solid support, a decrease
of the enzymatic activity relative to the free enzyme is often Materials and methods
2
7,28
observed in such systems.
Strong interactions between
Materials
enzymes and the host materials can be controlled by comple-
mentary organo-functionalization of the porous network, result-
Tetraethyl orthosilicate (TEOS, Aldrich, Z99%), phenyltri-
ethoxysilane (PhTES, Aldrich, 498%), cetyltrimethylammonium
p-toluenesulfonate (CTATos, Sigma), triethanolamine (TEA,
Aldrich, 98%), (3-chloropropyl)triethoxysilane (CPTES, Fluka,
2
9–31
ing in different catalytic activity and loading capacities.
As
the functionalization of the mesoporous framework can be easily
modified, the organo-moieties can be adjusted to the require-
ments of the respective enzyme and the preferred attachment
reaction.
9
9
5%), diethyl ether (Et
9%), ethanol (EtOH, Aldrich, absolute), conc. hydrochloric
NO , Sigma,
9%), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC, Alfa
2
O, Sigma, 498%), sodium azide (Fluka,
acid (HCl, Aldrich, 37%), ammonium nitrate (NH
9
4
3
Furthermore, the application of mesoporous silica supports
allows for the adjustment of pore- and particle size, structural
arrangement and surface properties to optimize loading capa-
Aesar, 98%), 4-pentynoic acid (Fluka, 97%), tris(hydroxymethyl)-
aminomethane (TRIS, Sigma, 99.8%), 2-(N-morpholino)ethane-
sulfonic acid (MES, Sigma, 99.5%), carbonic anhydrase from
bovine erythrocytes (Sigma, Z95% (SDS-PAGE), specific activity
Z3500 W A units per mg protein, lyophilized powder), peroxidase
from horseradish (Sigma, Type VI-A, essentially salt-free,
lyophilized powder, 950–2000 units per mg solid (using ABTS),
Z250 units per mg solid (using pyrogallol)), (+)-sodium
L-ascorbate (Sigma, Z98%), copper(II) sulfate (Sigma, Z99.99%),
disodium hydrogen phosphate (Na HPO , Sigma, 499%), mono-
8
,32,33
city and enzyme activity.
Lee et al. have reviewed different
methods of covalent enzyme immobilization onto mesoporous
3
4
silica supports. Here, we use the flexibility of silica function-
alization for loading active enzymes and our newly developed
MSNs with large pores to immobilize enzymes while retaining
their activity. Additionally, we make use of the demonstrated
reusability of immobilized horseradish peroxidase in SBA-16 to
be able to study the catalytic behaviour and improved stability
3
5
2
4
over more than one cycle. Previous studies describe the immo-
bilization of horseradish peroxidase (HRP) onto the surface of
mesoporous silica nanoparticles via physical adsorption or
sodium hydrogen phosphate (NaH PO , Sigma, 499%), sodium
hydroxide (1 M, Sigma), dipotassium hydrogen phosphate
2
4
3
6,37 (K HPO , Sigma, 499%), monopotassium hydrogen phosphate
2
4
covalent binding by using the crosslinker glutaraldehyde.
(KH
2
PO
4
, Sigma, 499%), 4-nitrophenyl acetate (Sigma, esterase
Due to the fact that glutaraldehyde is toxic and rather unstable
3
8
substrate), guaiacol (Sigma, oxidation indicator, Z98.0%),
hydrogen peroxide (Sigma, 30 wt%). Unless otherwise noted,
all reagents were used without further purification. Bi-distilled
water from a Millipore system (Milli-Q Academic A10) was used
for all syntheses and purification steps.
in air, here we use a modified azide–alkyne click chemistry
approach established by Schlossbauer et al. to immobilize
3
9,40
enzymes into the LP-MSNs.
This method is biocompatible
and avoids unfavorable pore blocking. The same immobiliza-
tion technique is also used to covalently bind an essential
metalloenzyme, namely carbonic anhydrase, to the pore walls
of the mesoporous silica nanoparticles. The immobilization of
Methods
enzymes in general enables their already mentioned reusability For transmission electron microscopy, samples were investi-
4
1
and furthermore their long-term use in bioreactors.
gated with an FEI Titan 80-300 transmission electron micro-
Two different enzymes, namely the lyase carbonic anhydrase scope (TEM) operating at 300 kV with a high-angle annular dark
CA) and the peroxidase horseradish peroxidase (HRP), were field detector. A droplet of the diluted MSN solution in absolute
(
chosen for this study and were immobilized with an efficient ethanol was dried on a carbon-coated copper grid. The TEM, SEM
method into the newly developed azide-functionalized LP-MSNs. and STEM micrographs in Fig. 1a–c show template-extracted
A novel co-condensation synthesis approach was developed to mesoporous large pore silica particles (LP-MSN–N ) generated
3
generate organo-functionalized MSNs with large pores suitable with the above described synthesis procedure (details below).
for biocompatible coupling methods. To study the catalytic Nitrogen sorption measurements were performed on a Quanta-
activity of the immobilized enzymes, two suitable activity assays chrome Instruments NOVA 4000e and on an Autosorb iQ
were performed. In the case of carbonic anhydrase, the instrument. All samples with attached enzyme (15 mg each)
catalyzed hydrolysis of the substrate 4-nitrophenol acetate was were treated at 298 K (12 h in vacuo), whereas the sample
studied by following the increasing absorbance of yellow LP-MSN–N was heated to 393 K for 12 h in vacuo (10 mTorr) to
3
4-nitrophenol at 400 nm over time. For immobilized horse- outgas the samples before nitrogen sorption was measured at
radish peroxidase, the catalyzed oxidation of guaiacol (2-methoxy- 77 K. For calculations of pore sizes and volumes a non-local
phenol) was recorded at a wavelength of 470 nm over time. It was density functional theory (NLDFT) equilibrium model of nitro-
shown that the immobilized enzymes exhibited high stability gen on silica was used. The obtained physisorption data for
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LP-MSN–N
3
are shown in Fig. 1e (nitrogen sorption isotherm) was determined with Nanodrop. The alkyne-functionalized
and Fig. 1f (pore size distribution) and in Fig. 2a (isotherms) and carbonic anhydrase was used for click reactions without
Fig. 2b (pore size distribution) for LP-MSN–CA and LP-MSN–HRP. further purification.
Dynamic light scattering (DLS) measurements were performed on
Horseradish peroxidase (sp-HRP). Alkyne-functionalized
a Malvern Zetasizer-Nano instrument equipped with a 4 mW horseradish peroxidase was synthesized according to Gole et al.
42
À1
He–Ne laser (633 nm) and an avalanche photodiode. The hydro- as described above. The concentration of sp-HRP (1.32 mg mL
)
dynamic radius of the particles was determined by dynamic light was determined with UV-vis spectroscopy (calibration curve,
scattering in water. The data are shown in Fig. 1d. Thermogravi- Fig. S2, ESI†).
metric analysis of the bulk samples LP-MSN–N , LP-MSN–CA and
3
LP-MSN–HRP was performed on a Netzsch STA 440 C TG/DSC Synthesis of (3-azidopropyl) triethoxysilane
À1
with a heating rate of 10 K min in a stream of synthetic air of
(
3-Azidopropyl)triethoxysilane was synthesized according to a
À1
about 25 mL min . The mass was normalized to 100% at 130 1C
43
procedure published by Nakazawa et al. (3-Chloropropyl)-
for all samples and the resulting data are presented in Fig. 2c. All
IR measurements were performed on a Thermo Scientific Nicolet
iN10 IR Microscope in reflection–absorption mode with a liquid
nitrogen-cooled MCT-A detector. All spectra are background
corrected. UV-vis measurements were performed on a Perkin
Elmer Lambda 1050 spectrophotometer with a deuterium arc
lamp (UV region) and a tungsten filament (visible region). The
detector was an InGaAs integrating sphere detector. Time-based
enzyme activity assays were performed in macro PMMA-cuvettes
triethoxysilane (10 g, 41.7 mmol) and sodium azide (10 g,
154 mmol) were heated in anhydrous DMF (100 mL) under
inert gas conditions at 90 1C for 4 h. Low boiling materials were
removed from the mixture under reduced pressure. The crude
oily product was dissolved in diethyl ether, resulting in the
precipitation of salts, which were removed by filtration. Et
was removed in vacuo and the residual oil was distilled (95 1C).
3-Azidopropyl)triethoxysilane was obtained as colorless liquid
2
O
(
and used without further purification.
(path length 10 mm). For small sample amounts (2 mL of the
sample) a NanoDrop 2000c spectrometer from Thermo Scientific
Fisher was employed for UV-vis measurements. The absorbance
at 280 nm (mode A280) was used to determine carbonic anhydrase
concentrations.
Synthesis of azide functionalized large pore mesoporous silica
nanoparticles (LP-MSN–N )
3
Core–shell bi-functional (MSN–N3,in–Phout) MSNs were synthe-
sized through a delayed co-condensation approach based on
1
8
Buffer preparation
an adapted recipe reported in the literature. In brief, TEA
(
0.047 g, 0.32 mmol), CTATos (0.263 g, 0.58 mmol) and H
2
O
MES buffer (0.1 M, pH 6.0). 2-(N-Morpholino)ethanesulfonic
acid (MES, 9.76 g) was dissolved in water (500 mL). The pH was
adjusted to 6.0 using sodium hydroxide (1 M).
Sodium phosphate buffer (5 mM, pH 7.4). Monosodium
hydrogen phosphate (2.92 g) and disodium hydrogen phosphate
(13.7 g, 0.77 mmol) were mixed in a 100 mL round bottom flask
and stirred vigorously (1250 rpm) at 80 1C until the surfactant
solution became homogeneous. TEOS (1.71 g, 8.2 mmol) and
AzTES ((3-azidopropyl)triethoxysilane) (120 mL, 0.41 mmol;
5
mol%) were mixed and added to the surfactant solution and
the mixture was stirred (1250 rpm) at 80 1C for 1 h. Afterwards,
01 mg of TEOS (0.96 mmol) was separated into 4 aliquots and
(7.74 g) were dissolved in 10 L of water (5 mM). The pH was fine-
adjusted to 7.4 with NaOH or HCl using a pH-meter.
Potassium phosphate buffer (0.2 M, pH 7.0). Mono-
potassium hydrogen phosphate (13.61 g) and dipotassium
hydrogen phosphate (17.40 g) were dissolved in water (500 mL).
The pH was fine-adjusted to 7.0 with NaOH or HCl using a
pH-meter.
TRIS buffer (0.1 M, pH 8.0). Tris(hydroxymethyl)amino-
methane (TRIS, 6.075 g) was dissolved in water (500 mL). The
pH was adjusted to 8.0 using HCl (1 M).
2
added to the mixture every 3 minutes. The solution was further
stirred for 1 h at 80 1C. A mixture of TEOS (20.1 mg, 0.096 mmol)
and PhTES (phenyltriethoxysilane) (22.9 mg, 0.096 mmol,
1
mol%) was then added to the solution to generate the outer
shell of the MSNs. The resulting solution was continuously
stirred at 80 1C for 1 h. The as-synthesized particles were collected
by centrifugation (7197g, 20 min) and were subsequently template
extracted (see below).
Synthesis of alkyne-modified enzymes (sp-enzymes)
Template removal from LP-MSN–N
The organic template was removed via extraction from
. Thus, 1 g of the as-synthesized particles was heated
3
Carbonic anhydrase (sp-CA). To a freshly prepared, cooled
À1
solution (4 1C) of carbonic anhydrase (40 mg, M
w
= 29000 g mol
,
1
.38 mmol) in MES buffer (pH 6, 18.7 mL, 0.1 M), an aqueous LP-MSN–N
3
solution (1 mL) of 4-pentynoic acid (0.1 M, 9.81 mg, 0.1 mmol) in an ethanolic solution (150 mL) containing 3 g ammonium
was added. The resulting mixture was vortexed for 2 min and nitrate at 90 1C under reflux for 1 h, cooled and collected by
then stored at 4 1C for 15 min. Subsequently, EDC (11.5 mg, centrifugation (7197g, 20 min) and extracted by a second reflux
0.07 mmol) was added to the solution. The reaction was vortexed at 90 1C using a 2 M HCl/ethanolic solution (150 mL) for 1 h.
again for 2 min and then kept at 4 1C for 4 h without stirring. Afterwards, samples were washed in the sequence EtOH, H O,
2
Afterwards, the obtained solution was dialyzed in a cold room at EtOH (100 mL per wash) and were finally kept in 20 mL EtOH.
4
1C against sodium phosphate buffer (5 mM, pH 7.4, 10 L) for a Particles were collected by centrifugation (7197g, 20 min) after
À1
period of 24 h. The concentration of sp-CA (3.027 mg mL
)
each template extraction and washing step.
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Immobilization of alkyne-functionalized carbonic anhydrase in the published protocol of Maehly and Chance was used. All
LP-MSN–N (LP-MSN–CA) substrate solutions and potassium phosphate buffer (pH 7.0,
.2 M) were freshly prepared. 5 mg LP-MSN–HRP was redispersed
3
0
An ethanolic suspension containing 50 mg LP-MSN–N
3
was centri-
in 100 mL H O and 3 mL buffer as well as 50 mL guaiacol solution
2
fuged (10 min, 7197g), washed with 15 mL sodium phosphate
buffer (0.2 M, pH 7.2) and re-dispersed in 2.5 mL sodium phos-
phate buffer as well as 2 mL water. The suspension was vortexed
for 2 minutes and put into an ultrasonic bath for 10 minutes to
agglomerates. Subsequently, the buffered solution of sp-CA (con-
taining 9 mg, 0.3 mmol sp-CA) was added and the resulting mixture
was stirred for 30 minutes at room temperature. In the meantime,
sodium ascorbate (1 mg, 5.05 mmol) was added to a solution of
(0.5 mM, 2.8 mL in 50 mL water) added and mixed. Subsequently,
2
0 mL H (12.3 mM) was added, the cuvette once inverted and
2 2
O
the time-dependent formation of the coloured product deter-
mined by UV-VIS spectroscopy (470 nm, 100 s, intervals of 1 s). All
spectra were background corrected.
Cu(II)SO
0 min at ambient temperature. 12 mL of this freshly prepared
solution was added to the reaction mixture containing LP-MSN–N
and sp-CA to catalyze the covalent attachment of the enzyme via
click chemistry. The resulting suspension was shaken (600 rpm) for
4
(1.25 mg, 1 mmol) dissolved in 5 mL water and stirred for Results and discussion
1
Synthesis of large pore MSNs
3
For the immobilization of alkyne-functionalized enzymes by
a bio-orthogonal click chemistry approach, core–shell meso-
porous silica nanoparticles with large pores and azide groups
on the internal and phenyl groups on the external surface
30 minutes at room temperature. Subsequently, the particles were
collected by centrifugation (10 min, 5500g, 15 1C) and re-dispersed
in 30 mL water. This washing procedure was repeated three times.
LP-MSN–CA were obtained as colloidal solution in water and were
stored at À20 1C until further use.
(
LP-MSN–N ) were synthesized using the delayed co-condensation
3
procedure developed in our group following a modified protocol
18,46
from Zhang et al.
In brief, a delayed co-condensation approach was chosen
to introduce small amounts (5 mol% of the original TEOS
amount) of azide-functionality into the inside of the mesoporous
framework of the LP-MSNs and to obtain a homogeneous dis-
tribution of organic moieties within the silica material to ensure
immobilization of the enzymes only inside the pores of the
particles. Higher degrees of organo-modification can result in a
reduction of the pore diameter and would thus impede the
diffusion of enzyme molecules into the mesoporous channels
and increase the likelihood of pore blocking. For the introduc-
tion of azide-moieties in the core, the silica source tetraethyl
orthosilicate (TEOS) was co-condensed with (3-azidopropyl)tri-
ethoxysilane (AzTES, 5 mol% of total silane content) while
PhTES (1 mol% of total silane content) and TEOS were used
to generate phenyl groups on the external surface. A shell of
pure TEOS was condensed in-between to create an interlayer
between the two different functional groups. Additionally, we
found that the introduction of organo-moieties in the case of
azide-silanes has no negative effect on the formation of the
mesoporous structure, whereas amine-functionalities easily
disrupt the large-pore nanoparticle formation. TEM and SEM
micrographs (Fig. 1a–c) of the resulting core–shell particles
Horseradish peroxidase (LP-MSN–HRP)
Sp-Horseradish peroxidase was immobilized onto LP-MSN–N
via a click chemistry approach according to the synthesis
procedure described above for sp-CA. An ethanolic suspension
3
containing 50 mg LP-MSN–N was centrifuged (10 min, 7197g),
3
washed and re-dispersed as described above. Afterwards, the
buffered solution of sp-HRP (containing 9 mg, 0.2 mmol sp-HRP)
was added and the mixture was stirred for 30 minutes at room
temperature before the freshly prepared catalyst solution con-
sisting of sodium ascorbate and Cu(II)SO (see above) was added.
4
LP-MSN–HRP nanoparticles were obtained as colloidal solution
in water after several washing steps.
Catalytic activity determination of LP-MSN–CA
The catalytic activity of LP-MSN–CA was determined by an adapted
44
method published by Shank et al. Fresh solutions of 4-nitrophenyl
acetate (NPA, 10 mM, 18.1 mg, 0.1 mmol in 10 mL EtOH) and TRIS
buffer (50 mM, pH 8) were prepared shortly before the experiment.
500 mL of an aqueous suspension of LP-MSN–CA (containing 5 mg
particles and therefore 0.71 mg carbonic anhydrase, calculated from
TGA-data) were mixed in macro PMMA-cuvettes with 400 mL TRIS
buffer. Subsequently, 100 mL NPA-solution were added quickly
before the time-dependent hydrolysis of the substrate was deter-
mined by measuring the absorbance of the formed 4-nitrophenol
(MSN–N3,in–Phout) show a stellate morphology and a conical-
like pore structure.
Transmission electron microscopy images show spherically
shaped nanoparticles with a fairly homogenous particle size
distribution within a size range of 60–90 nm (Fig. 1a). The large
pores are present in each LP-MSN and evenly distributed over
the entire nanoparticle. The particle size distribution data are
in good accordance with the values obtained by dynamic light
scattering, exhibiting a maximum at 150 nm (Fig. 1d). We
explain the moderately increased apparent size obtained by
DLS measurements compared to the TEM measurements
(
400 nm, 120 s and intervals of 1 s). All spectra were background
corrected. After a finished cycle, the assay mixture was centrifuged
10 min, 16 900g, 15 1C) and washed with 1 mL water. The washing
(
procedure was repeated three times before the particles were
redispersed in 500 mL water and used for the next cycle.
Catalytic activity determination of LP-MSN–HRP
To determine the catalytic activity of immobilized horseradish with the larger hydrodynamic diameter of the LP-MSN–N
3
in
peroxidase (LP-MSN–HRP) a slightly modified assay following comparison to their real size – possibly weak agglomeration is
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Fig. 1 Characterization of LP-MSN–N
3
. (a) Transmission electron microscopy (TEM) image, (b) scanning electron microscopy (SEM) image and (c)
scanning transmission electron microscopy (STEM) image of template-extracted LP-MSN–N
3
, (d) dynamic light scattering (DLS) data showing a narrow
size distribution, (e) nitrogen sorption isotherm and (f) pore size distribution obtained from nitrogen sorption measurement for LP-MSN–N
3
.
3
48
also present. The mesoporous framework is well-defined and 4.0 Â 4.4 Â 6.8 nm ), were immobilized in the mesoporous
clearly present in all particles (Fig. 1a–c). The pore sizes are in a nanoparticles. Specifically, a modified click chemistry approach
range of about 6–10 nm (deduced from TEM measurements) and was used to immobilize the enzymes within the mesoporous
consistent with the wide pore size distribution of about 2.5–12 nm framework of our newly generated LP-MSNs, which required
obtained from nitrogen sorption measurements (Fig. 1f). The prior alkyne functionalization of the enzyme (for details see
3
9,42
corresponding sorption data show a hybrid isotherm of type IIb Methods section and Scheme 1).
The latter was achieved by
combined with a certain fraction of type IV. The narrow hysteresis the EDC-activated attachment of 4-pentynoic acid to the amine
can be characterized as an H3 hysteresis loop that results from moieties of CA or HRP, respectively (Scheme 1). The reaction
aggregation and inter-granular pores and thus from a significant took place in MES buffer (pH 6.0) at 4 1C.
degree of textural porosity, indicated by the long tail in the pore size
distribution (Fig. 1f). Monolayer formation of the adsorbate can be incubated with the alkyne-functionalized enzymes for 30 minutes
observed between p/p = 0 and 0.2, followed by multilayer adsorp- to ensure their diffusion into the large mesopores before the click
tion over a broad range. The apparent lack of saturation (pore reaction catalysts were added to the reaction mixture, in order
filling) at high p/p could be due to a broad distribution of textural to avoid pore blocking (for details see Methods section).
3
In a second synthesis step, the LP-MSN–N particles were
0
0
porosity between the dried particles. The inter-particle voids, which Subsequently, a click reaction was performed according to a slightly
are visible in the micrographs (Fig. 1a–c), are within a size-range of modified procedure published by Himo et al., employing sodium
5
0
1
0–30 nm. Summarizing, we obtained LP-MSNs exhibiting a pore ascorbate as reducing agent for Cu(II) (CuSO
4
) (Scheme 2).
size in the range of mesoporous materials (Fig. 1f, maximum
3
À1
around 7 nm), with a pore volume of 1.6 cm g and a BET
2
À1
surface area of A
was carried out and verified the presence of azide-moieties within
the pore system of LP-MSN–N (Fig. S1, ESI†).
s
= 670 m g . In addition, infrared spectroscopy
3
Immobilization of alkyne-functionalized carbonic anhydrase
Scheme 1 Alkyne-functionalization of enzymes. EDC activated attach-
ment of 4-pentynoic acid to the respective enzyme (carbonic anhydrase
or horseradish peroxidase; here shown with bovine carbonic anhydrase II).
The reaction was carried out in MES buffer (pH 6.0) at 4 1C. The accession
and horseradish peroxidase onto LP-MSN–N
chemistry approach
3
via a click
Two different enzymes, namely carbonic anhydrase (CA, 30 000 Da,
number from the protein data bank (PDB) for the X-ray structure of BCAII
3
47
49
4.0 Â 4.2 Â 5.5 nm ) and horseradish peroxidase (HRP, 44 000 Da, is 1V9E.
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probe the pore. We assume that the decrease in pore volume in
case of the sample LP-MSN–CA is stronger than in LP-MSN–
HRP because a larger amount of sp-CA could be incorporated
into the mesoporous framework of our nanoparticles. These
results are in good accordance with the thermogravimetric
analysis (TGA) data (Fig. 1c and Table 1 for relative mass loss),
showing that more carbonic anhydrase than horseradish per-
oxidase was immobilized in the LP-MSNs. The higher degree of
immobilization of CA as compared to HRP is attributed to its
smaller dimension; the enzyme can diffuse more easily into the
mesopores without having as much contact with the pore walls
3
Scheme 2 Covalent attachment of sp-enzymes into LP-MSN–N . Scheme
for protein immobilization in LP-MSNs using click chemistry. Cu(II) was
reduced with sodium ascorbate resulting in Cu(I), the actual catalyst.
The nanoparticles were washed several times with water in and the attached organic moieties, while HRP is bigger and
order to remove excess and unbound enzyme molecules. thus has a higher likelihood to touch the pore walls, bind to
All obtained nitrogen sorption data show isotherms that them and block access to the pore for additional molecules.
appear as hybrids between type II and type IV isotherms, with a From the additional mass loss of 11.5% for the sample
narrow H3 hysteresis loop. After the attachment of sp-enzymes LP-MSN–CA compared to LP-MSN–N , the amount of attached
3
the calculated BET-surface decreased significantly (Fig. 2a and enzyme can be estimated to be 4.7 mmol per g silica (for calcu-
Table 1 respectively). This can be attributed to the uptake and lation see Calculation A1 in the ESI†), which corresponds to
covalent immobilization of the enzyme molecules within the 141 mg carbonic anhydrase per g silica support. The amount
large mesopores of the nanoparticles and the resulting partial was additionally determined via Bradford assay, resulting in
pore filling. Accordingly, the pore volume decreased likewise 99 mg CA per g silica (see ESI,† Fig. S10). This deviation of almost
(Fig. 2b). A strong reduction of the very small pore sizes can be 28% is attributed to additional components in the channels of the
observed. We attribute this to the conical-like shape of the mesoporous silica that contribute to weight changes during TGA
pores: enzymes that diffuse entirely into the pore, block the measurements. The determined amount indicates that 0.5% of
small end of it and thereby the parts of the pores representing all azide-moieties have reacted with sp-CA, as the amount of
small pore sizes. Carbonic anhydrase shows a stronger reduc- azide-moieties was estimated to 1.02 mmol per g silica. From
tion of the smaller pore sizes than horseradish peroxidase, the additional mass loss of 9.4% for the sample LP-MSN–HRP
possibly due to their different shape: the shape of CA is close to compared to the azide-functionalized LP-MSNs, the amount
a square and thus, when it gets stuck in a pore due to size of immobilized horseradish peroxidase can be estimated to
limitations, it may block the entire pore uniformly in all direc- 2.6 mmol per g silica (see ESI,† Calculation A1), or to 114 mg
tions. HRP is rod-shaped, if it blocks a pore there is space on its HRP per g silica support. The amount determined with the
sides that allows for BET measurements to still access and additionally performed Bradford assay (see ESI,† Fig. S11)
3
Table 1 Nitrogen physisorption and thermogravimetric analysis data of LP-MSN–N and LP-MSN–enzyme
2
À1
a
3
À1
b
Sample
BET surface area [m g
]
Pore size range [nm]
Pore volume [cm g
]
Relative mass loss [%]
LP-MSN–N
LP-MSN–CA
LP-MSN–HRP
3
670
389
423
2.5–12
5.5–12
5.3–12
1.6
1.2
1.4
3
9.2 (7.7% for N -moieties)
20.7
18.6
a
b
DFT pore size range refers to the main range of the pore size distribution. Relative mass loss obtained by TGA; all curves are normalized
to 130 1C.
3 3
Fig. 2 Nitrogen sorption data of LP-MSN–N with immobilized enzymes. (a) Isotherm of LP-MSN–N (black), LP-MSN–CA (purple) and LP-MSN–HRP
(
cyan). (b) Pore size distribution for LP-MSN–N
3
(black), LP-MSN–CA (purple) and LP-MSN–HRP (cyan). (c) Thermogravimetric analysis data of LP-MSN–N
3
(black), LP-MSN–HRP (cyan) and LP-MSN–CA (purple). All TGA curves are normalized to 100% at 130 1C.
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differs very little from the above TGA results and corresponds to pellet size and accordingly a decrease in absorbance and slope
03 mg HRP per g silica. This indicates that a smaller amount (Fig. 3a). The self-hydrolysis of 4-NPA was measured separately
1
of sp-Horseradish peroxidase molecules was able to diffuse (data shown in Fig. S6, ESI†). The self-hydrolysis reaction data were
into the large mesopores and reacted with 0.25% of all azide- subtracted from the raw data of the enzyme-catalyzed hydrolysis.
moieties of LP-MSN–N . A probable reason for this is the even In Fig. 3a and Fig. S7 (ESI†) the CA activity corrected for self-
3
larger molecular dimension of horseradish peroxidase (4.0 Â hydrolysis is shown. For quantitative determination of the reaction
3
48
4
.4 Â 6.8 nm ), resulting in partial blocking of the pores by rate, the slope of one cycle at a reduced substrate concentration
immobilized horseradish peroxidase. All nitrogen sorption data (1 mM NPA) was converted into concentration vs. time (Fig. S7,
are supported by the corresponding thermogravimetric analysis ESI†) by using Lambert–Beer’s law (e = 16 300 M
À1
À1 51
cm ).
and the calculated relative mass losses that are summarized According to Michaelis–Menten kinetics in the limit of low sub-
in Table 1. As further proof for the successful attachment of strate concentrations, the kinetic constant kcat/Km was determined
À1 À1
sp-enzymes via click chemistry, vibrational spectroscopy was to be 74 M
s . There are large differences in the kinetic
52
performed with the respective LP-MSN–enzyme samples (see constants for different CAs reported in the literature. Therefore,
Fig. S3 for CA and S4 for HRP, ESI†). The symmetric and it is difficult to compare our result for immobilized bovine
asymmetric CH
in the range of 2960–2871 cm in the spectrum of LP-MSN–CA
2
stretching vibrations of the enzyme appear carbonic anhydrase to literature values.
À1
The resulting activity of immobilized carbonic anhydrase was
and sp-CA and of LP-MSN–HRP and sp-HRP, respectively. The also determined in international units IU, which is described in
À1
peak at 2105 cm can be attributed to the asymmetric N
3
the ESI.† The expressed activity of the biocatalyst LP-MSN–CA
stretching vibration of LP-MSN–N and can still be observed in results in 8.4 units (per 5 mg MSNs) i.e. 1680 units per g support.
3
LP-MSN–enzyme due to the small fraction of reacted (clicked) The corresponding amount of free enzyme shows an activity of
À1
azide-moieties. At 1658 cm (dashed line) the CQO stretching 85.6 units and therefore performs one order of magnitude faster
vibration appears in the spectrum of LP-MSN–CA/HRP, which is than the immobilized enzyme, which might be attributed to
attributed to the amide bonds of the attached enzymes. The partial blocking of the enzyme due to the statistical attachment
À1
vibration at 1530 cm (second dashed line) is only visible in the of the enzyme molecules inside the pores. All in all, it could be
spectra of sp-enzyme and LP-MSN–enzyme and represents shown that carbonic anhydrase was successfully immobilized
the N–H bending vibration of the peptide bonds of the enzyme. inside the large pores of our MSNs and remained there stable
À1
The peak at 1627 cm in the spectrum of LP-MSN–N indicates the and active.
3
À1
presence of traces of water within the sample. Below 1300 cm
,
Horseradish peroxidase activity determination
vibrations of the silica framework appear with strong intensity.
Summarizing, the results obtained by nitrogen sorption, thermo- In order to determine the catalytic activity of the MSN-bound
gravimetric analysis and vibrational spectroscopy all support the HRP molecules, an assay involving the oxidation of guaiacol
4
5
successful immobilization of carbonic anhydrase and horseradish was performed. Thereby, the catalyzed oxidation of guaiacol
peroxidase within the porous network of LP-MSN–N . In the was measured by recording the absorption of the produced
following we address the catalytic properties of the immobi- tetraguaiacol at 470 nm with UV-vis spectroscopy (Fig. 3b, for
3
lized enzymes.
experimental details see Methods section and Fig. S8, ESI†).
The remaining stability and activity of the immobilized enzyme
could be shown over several cycles just as well as in the case of
carbonic anhydrase. To determine the reaction rate, the slope in
Catalytic activity determination of immobilized carbonic
anhydrase
To characterize the properties of immobilized CA, the catalytic the linear region (0 s–15 s) of the first cycle’s graph (gray graph)
activity of the hydrolysis of 4-nitrophenylacetate (NPA) was was used and converted into concentration vs. time (Fig. S9,
À1
À1
studied colorimetrically by measuring the absorbance of the ESI†) using Lambert-Beer’s law (with e = 26.6 mM cm from
4
5
newly formed yellow nitrophenol at 400 nm (Fig. 3a and Fig. S5, Maehly and Chance and d = 1 cm). The rate was calculated to
4
À1
À1
45
ESI†). To ascertain the robust attachment and stability of the be 2.5 Â 10 M cm according to Maehly and Chance. In
4
5
5
À1
À1
enzyme inside the pores, multiple cycles were performed. After comparison to the literature value of 3 Â 10 M cm , our
cycle 1, the reaction mixture was transferred to small Eppen- immobilized enzyme performs slower by about one order of
dorf tubes and centrifuged for 5 min (15 1C, 16 900g). The pellet magnitude than the free corresponding sp-enzyme. The result-
was redispersed in water and centrifuged again. This washing ing activity of immobilized horseradish peroxidase (definitions
step was conducted two times to remove (un)reacted substrate. of IU at the end of the ESI†) is 0.86 units (per 0.16 mg MSN) i.e.
Afterwards, the washed particles were redispersed in water and 5343 units per g support. The corresponding amount of free
fresh substrate was added for the next cycle. In total eight such enzyme shows an activity of 3.7 units, resulting in an only
cycles with intermediate washing steps were performed (Fig. 3a). fourfold better performance than our immobilized HRP.
The time-based measurements show a successful conversion of
Note that the enzyme concentration used in the calculations
4-NPA to nitrophenol in all cycles, proving the continuous is the upper limit. Due to a necessary centrifugation step before
activity and stability of the immobilized carbonic anhydrase. the activity assay a small loss of particles and therefore enzyme
Due to the washing steps in between the cycles, a certain loss of is inevitable, thus reducing the enzyme concentration. Since
particles and therefore also enzyme can be seen by a decrease in the enzyme concentration is inversely proportional to the
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Fig. 3 Activity determination for LP-MSN–CA and LP-MSN–HRP as well as for free versus alkyne-functionalized enzymes. (a) UV-vis absorption
measurement of catalyzed hydrolysis of the substrate 4-nitrophenol acetate (NPA) by LP-MSN–CA within a period of 120 s (interval 1 s) at 400 nm with
1
0 mM NPA in TRIS buffer (pH 8, 50 mM) over eight cycles. (b) Activity determination of LP-MSN–HRP by measuring the absorbance at 470 nm of
emerging oxidized guaiacol (2-methoxyphenol) in potassium phosphate buffer (pH 7, 0.2 M) for 100 s (interval 1 s) over eight catalytic cycles. The
enzyme’s reaction rate was determined from the slope of the first cycle’s linear region (gray curve, from 0 s–15 s). (c) Catalytic activity of native versus
functionalized carbonic anhydrase (assay described in (a)). Comparison of catalytic activity of native (free) CA (purple line Æ SD light purple area) versus
functionalized CA (sp-CA) (cyan line Æ SD light cyan area). (d) Activity comparison of native versus functionalized horseradish peroxidase (assay described
in (b)). Activity determination of native (free) HRP (purple line Æ SD light purple area) versus sp-HRP (cyan line Æ SD light cyan area) by measuring the
absorbance of oxidized guaiacol at 470 nm for 100 s.
calculated reaction rate, our value of the calculated reaction functionalities can be efficiently introduced at controlled con-
rate represents a lower limit. Additionally, some horseradish centrations. This method further avoids additional grafting
peroxidase molecules could be partially hindered or blocked due steps that may be difficult with colloidal systems. Our synthesis
to the random attachment of the large enzymes into the rather yields mesoporous silica nanoparticles within a size range of
tightly matching pores; the active center might not always be about 100 nm exhibiting large pore sizes of up to 12 nm and
perfectly accessible for the substrates. This may explain the carrying azide-functionalities inside the pore system. This organo-
lower reaction rate in comparison to unbound HRP.
functionalization makes the LP-MSNs suitable for a biocompati-
To prove that alkyne-functionalisation of the enzymes does ble modification via click chemistry. We covalently immobilized
not impair their catalytic activity, the activity assays were the two enzymes carbonic anhydrase and horseradish peroxidase
3
performed with the native versus alkyne-functionalized enzymes. in the interior of the LP-MSN–N host.
3
47
In both cases (Fig. 3c and d) it can be shown that the catalytic
performance is maintained after enzyme-functionalization.
The lyase carbonic anhydrase (CA) (4.0 Â 4.2 Â 5.5 nm ) and
3 48
the peroxidase horseradish peroxidase (HRP) (4.0 Â 4.4 Â 6.8 nm )
were successfully modified with alkyne-linkers to enable grafting
through a Cu-catalyzed click reaction. The enzymes were allowed
to diffuse into the mesopores before the catalysts for the click
reaction were added to the reaction mixture, thus avoiding
Conclusion
We report a facile delayed co-condensation strategy for the unfavorable pore blocking and low catalytic activity. We could
synthesis of spatially segregated core–shell bi-functional large- demonstrate in this study that both enzymes are still highly
pore MSNs. This direct approach towards introducing organo- active in their immobilized state, and that they maintain high
moieties into the mesopores is highly beneficial since different activity during activity assays over several cycles.
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Overall, we present a spatially structured colloidal MSN- 11 H. Jin, H. Qiu, Y. Sakamoto, P. Shu, O. Terasaki and S. Che,
based system with large pores for the immobilization of active
biomolecules that holds promise as a versatile platform for
numerous applications.
Mesoporous Silicas by Self-Assembly of Lipid Molecules:
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1
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Conflicts of interest
1
1
The authors declare no competing financial interest.
Acknowledgements
Financial support from the German Science Foundation (DFG)
1
(Cluster Nanosystems Initiative Munich (NIM) and SFB 1032,
project B05) and the Center for NanoScience Munich (CeNS)
is gratefully acknowledged. We thank Dr Steffen Schmidt for
electron microscopy.
1
6 S. B. Hartono, W. Gu, F. Kleitz, J. Liu, L. He, A. P. J.
Middelberg, C. Yu, G. Q. Lu and S. Z. Qiao, Poly-l-lysine
Functionalized Large Pore Cubic Mesostructured Silica
Nanoparticles as Biocompatible Carriers for Gene Delivery,
ACS Nano, 2012, 6, 2104–2117.
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