Enzyme-immobilized metal-organic framework nanosheets as tandem catalysts for the generation of nitric oxide

Article abstract of DOI:10.1039/c8cc05068f

An enzyme-immobilized metal-organic framework (MOF) nanosheet system was developed as a tandem catalyst, which converted glucose into gluconic acid and H₂O₂, and sequentially the latter could be used to catalyze the oxidation of l-arginine to generate nitric oxide in the presence of porphyrinic MOFs as artificial enzymes under physiological pH, showing great potential in cancer depleting glucose for starving-like/gas therapy.

Full text of DOI:10.1039/c8cc05068f

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Enzyme-immobilized metal-organic framework nanosheets as
tandem catalysts for generation of nitric oxide†
Received 00th January 20xx,
Accepted 00th January 20xx
Pinghua Ling,a* Caihua Qian,a Feng Gaoa and Jianping Leib*
DOI: 10.XXXX/x0xx00000x
www.rsc.org/
An enzyme-immobilized metal-organic framework (MOF) powered by a lot of reaction
nanosheet system was developed as a tandem catalyst, which cascades facilitated by the synergistic protein catalysts via complex
converted glucose into glucose acid and H2O2, and sequentially the 35metabolic pathways. Studies have shown that coupling enzymatic
latter could catalyze the oxidation of L-arginine to generate nitric catalysts and molecular catalysts or nanomaterials could construct
oxide in the presence of porphyrinic MOF as artificial enzyme tandem catalytic systems.16 So, it is interesting in designing a
under physiological pH, showing great potential in cancer starving- synergistic reaction system with enzymatic catalysts and
5
like/gas therapy.
nanomaterials to use under physiological pH and room temperature
40conditions.
Nitric oxide (NO) as a star molecule is an important endogenous
signaling molecule and plays a pivotal role in physiological and
Immobilizing the enzyme on the common platform support can
offer a plausible pathway. Recently, many conventional solid, such
as zeolites, and mesoporous silica,17 have been investigated to
immobilize enzymes. Nevertheless, the lack of specific interactions
45between these materials and enzymes molecules results in loss of
activity upon reuse.17a,17b,18 Metal−organic frameworks (MOFs), a
new class porous material, have been reported as enzyme mimetics
or as a support for enzyme immobilization.19 2D MOFs, a newly
developed material and a new member of the 2D material family,
50have received great attention.20 Compared to traditional 3D bulk
MOFs, 2D MOFs possess some advantages including large surface
area, more accessible active sites on their surfaces and thickness of
sub-10 nm.20b,20c 2D MOFs have been reported in versatile
applications, such as catalysis,20c,21 sensing,22 gas separation.20a,20b
55Inspired by the unique physical and chemical properties of 2D MOFs,
more and more attentions have been paid to synthesize and
explore the function of 2D MOFs. Herein, 2D MOFs, Co-TCPP(Fe)
nanosheets (Co-FeMOF) were chosen as both enzymatic mimic to
10pathological processes.1 Recently, NO has been used in the field of
cancer therapy. Furthermore, high NO content not only kills the
cancerous cell but also could enhance the efficacy of therapy.2
Inversely, low NO content may probably promote the progression
of disease.3 So it is necessary to develop activatable NO donors for
15the sustained NO release. L-Arginine (L-Arg) is a natural NO donor
and can continuously release NO in the presence of inducible NO
synthase (iNOS).4 Previous studies also indicate H2O2 could oxidize
L-Arg to NO.5 It is expected that L-Arg in the H2O2-rich tumor cells
could generate a large amount of NO. Thus, combining the NO-
20releasing materials and NO-generating catalysts with biomimic
capacity is a possible solution to form NO.
The artificial enzymes or biomimetic systems have been studied
for decades,6 including iron oxide,7 gold,8 copper9 nanoparticles,
Cu2+-modified carbon dots, carbon nitride nanoparticles or
25graphene oxide nanoparticles10 and prussian blue nanoparticles.11
Due to their unique characteristics relative to natural enzymes,
artificial enzymes have been extensively explored for different
applications, such as in bioanalysis,12 molecular delivery,13
bioimaging,14 and biomedicine.15 In contrast, the biological systems
30are complex chemical transformations rather than conventional
chemical reactions under mild conditions, such as physiological pH,
atmospheric pressure and aqueous solution. The processes are
catalyze the oxidation reaction and
a support for enzyme
60immobilization to establish a tandem catalysts system.
FeTCPP, an iron porphyrin species, could catalyze the oxidation
reaction of L-Arg by H2O2 to form citrulline and NO.5b However, it
generally undergoes molecular aggregation and oxidative
destruction, so it limits its application in many fields. Hydrophilic
65iron porphyrin derivatives immobilized on the resin could be used
for the oxidation of L-Arg, but the system needs
a high
concentration of H2O2.5c In order to achieve the artificial enzyme
system for tandem catalysis and local generation of NO, we
immobilized glucose oxidase (GOD) on the surface of Co-FeMOF
70which was prepared by Co as node and FeTCPP as linker (Scheme 1).
Glucose could be oxidized into glucose acid and H2O2 by GOD. On
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the other hand, FeTCPP could catalyze the oxidation reaction of L- transmission electron microscopy (TEM). The SEM result reveals the
Arg for generation of NO. The strategy is expected to produce H2O2 Co-FeMOF with sheet-like morphology and hundreds of
locally from endogenous glucose for the subsequent Co-FeMOF nanometers (Fig. 1B). TEM image and SAED (Fig. 1C) show a well-
catalytic oxidation of L-Arg to generate NO species.
30dispersed and well-defined ultrathin sheet-like structures and
5
In this way, this complex conjugate can be as a tandem catalyst polycrystalline nature. Compared with Co-FeMOF, the surface of
to enable the continuous generation of NO from physiologically GOD@Co-FeMOF displays some agglomerates on the surface (Fig.
abundant glucose and L-Arg, and demonstrate that the artificial 1D), indicating the GOD was loaded on the surface of Co-FeMOF.
enzyme system can be mixed with serum to generate NO. The Simultaneously, 1.0 mg/mL Co-FeMOF is optically equivalent to 0.25
tandem catalysis, GOD@Co-FeMOF, has a great potential in the 35mg/mL free FeTCPP (Fig. S1). According to the reduced mass in Fe
10cancer starving-like/gas therapy.
element, the GOD content is determined to be 24% in the
GOD@Co-FeMOF (Table S1 and S2).
In order to further investigate the functionalization of Co-
FeMOF with GOD, the UV-vis absorption spectra and the zeta
40potential were employed. As shown in Fig. S2A, the maximum
absorption of GOD at 278 nm (curve a), which is the characteristic
peak of GOD (Fig. S3). Compared with FeTCPP (curve b), the
maximum Soret absorption of Co-FeMOF (curve c) shifts from 412
nm to ∼427 nm, which maybe contribute to the hydrophobic
45nature of the octahedral cavity and the sensitivity of the Soret band
to the dielectric constant of the solvent.23 From the UV-vis
absorption spectrum of GOD@Co-FeMOF (curve d), a new Soret
absorption appeared at 278 nm in comparison with that of pure Co-
FeMOF, illustrating the successful loading of the GOD protein on
50the surface of Co-FeMOF. Zeta potentials were also investigated
(Fig. S2B). The results show that Co-FeMOF has a more positive
potential than FeTCPP, mainly due to the FeTCPP self-assembled
into Co-FeMOF with Co3+ and the uncoordinated carboxyl group on
the surface of the Co-FeMOF. The zeta potential of GOD@Co-
55FeMOF is slightly more negative than that of Co-FeMOF, indicating
the successful functionalization of Co-FeMOF with GOD.
Scheme 1 Schematic illustration of (A) preparation of GOD@Co-FeMOF
composite and (B) the tandem catalysis strategy of toward L-Arg oxidation
to produce NO.
15
In this work, the 2D MOFs, Co-FeMOF, was synthesized with Co
as node and FeTCPP as linker. Powder X-ray diffraction (PXRD) was
employed to investigate the polycrystalline feature of Co-FeMOF
(Fig. 1A). The result depicts that the peaks at 7.6°, 8.8°, and 17°,
which were indexed as (110), (002), and (004), respectively,
20indicating the successful assembly of Co-FeMOF.20c,21 The
morphology of Co-FeMOF was characterized by the scanning
electron microscopy (SEM), selected area electron diffraction
pattern (SAED) and
To examine the catalytic oxidation characteristics of Co-FeMOF,
L-Arg oxidation reactions were conducted by dispersing the FeTCPP,
Co-FeMOF and GOD@Co-FeMOF in a pH 7.4 PBS buffer with 20 mM
60L-Arg, along with 5.0 mM H2O2 as the oxidant and the production
was identified by NO probe, 3-amino,4-aminomethyl1-2′,7′-
difluorescein, diacetate (DAF-FM DA). It has been documented that
NO can react with DAF-FM DA to form benzotriazole derivative with
fluorescence emission. The fluorescence (FL) spectra were
65monitored at different conditions (Fig. 2), and the intensity increase
of the emission peak at 515 nm corresponded NO generation. As
shown in Fig. 2A, DAF-FM DA was mixed with H2O2 (curve a), L-Arg
(curve b), glucose (curve c), FeTCPP (curve d), Co-FeMOF (curve e)
and GOD@Co-FeMOF (curve f), individually, the benzotriazole
70derivative demonstrated none curve peaking at 515 nm. When DAF-
FM DA was allowed to react with the mixture L-Arg and H2O2, the
emission profile was increased at 515 nm (curve g), indicating H2O2
can oxidize L-Arg to produce NO.5,24 Meanwhile, upon incubation
with the mixture of FeTCPP, L-Arg and H2O2 (curve h), the mixture
75of GOD@Co-FeMOF, L-Arg and H2O2 (curve i) and the mixture of Co-
FeMOF, L-Arg and H2O2 (curve j) for 25 min, respectively, the DAF-
FM DA demonstrated a significant curve peaking at 515 nm.
Furthermore, with the equivalent amount of FeTCPP, the Co-FeMOF
catalysts show a higher activity, which could be attributed to
80FeTCPP with the monomeric molecular structure in Co-FeMOF. In
order to verify the catalysis is derived from FeTCPP, the FL of Co-
MOF which is prepared with Co and TCPP (nonmetallic porphyrin)
25Fig. 1 (A) Powder X-ray diffraction patterns, (B) SEM, and (C) TEM images of
Co-FeMOF. Inset: SAED pattern. (D) SEM image of GOD@Co-FeMOF.
2 | J. Name., 2012, 00, 1-3
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mixed with 20 mM L-Arg, 5.0 mM H2O2 and 5.0 μM DAF-FM DA was
studied (Fig. S4). Compared with Co-MOF, Co-FeMOF exhibits
(curve c) show negligible fluorescence intensity changes, while the
catalytic rate of the mixture of H2O2 and L-Arg increased (Fig.2D,
higher catalytic activity, suggesting Co in the MOF has not catalytic curve a) with the time increasing. Comparing with the mixture of
GOD, glucose and L-Arg (Fig.2D, curve b), the mixture of GOD@Co-
45FeMOF, glucose and L-Arg (curve c) shows a larger catalytic rate,
which is consistent with the FL results (Fig. 2A). In this system,
GOD@Co-FeMOF could oxidize glucose to yield H2O2, then the
produced H2O2 could oxidize L-Arg to generate NO species and a
byproduct of L-citrulline via the following two-steps (Fig. S6):
activity toward L-Arg oxidation, which is consistent with the result
of ICP-OES (Table S2). To study the activity of GOD@Co-FeMOF, the
kinetics and activity profile of the enzymatic reaction were studied
(Fig. 2B and 2C). Compared with FeTCPP (Fig. 2B, curve a), Co-
FeMOF (curve b) shows a higher catalytic rate, because FeTCPP as
linker in Co-FeMOF avoided the formation of bridged μ-oxide
5
10dimers (hindered access to catalytic sites), intermolecular self-
oxidation and oxidative self-degradation.5b,25 After conjugation with
GOD, the catalytic rate of GOD@Co-FeMOF (Fig. 2B, curve c) shows
GOD
(1)
(2)
Glucose + O2
Gluconic acid + H2O2
L-Citrulline + NO +H2O
Co-FeMOF
L-Arginine + H2O2
50
a
slower catalytic rate than the Co-FeMOF, indicating GOD
In order to further understand the impact of GOD@Co-FeMOF
in the system, the kinetics and activity profile of GOD@Co-FeMOF
with different concentration were studied (Fig. S7). The results
show that the catalytic activity increases with the increasing of both
immobilized on nanosheets surface shields part of the catalytic sites.
15Based on GOD-catalyzed decomposition reaction of glucose, the
result shows that the generated H2O2 concentrations quickly reach
a plateau within only 30 min (Fig. S5A), suggesting the high catalytic
efficiency of GOD. Fig. S5B shows GOD still keeps its activity after
conjugating on the surface of Co-FeMOF, owing to the process of
20immobilizing is environmental friendly.21c After 30 min, the
generated H2O2 concentrations reach a plateau.
55the concentration of the GOD@Co-FeMOF and the reaction time,
and reaches a plateau after the addition of 1.0 mg/mL GOD@Co-
FeMOF. The impact of glucose concentrations in the system was
measured by monitoring the time-dependent fluorescence intensity
of benzotriazole derivative at 515 nm, which shows the catalytic
60activity increased with the glucose concentration increasing (Fig. S8)
The feasibility of the tandem catalysis was investigated by
conducting the kinetics and activity profile of GOD@Co-FeMOF with and the estimated Km of glucose is 4.6 mM according to the
Lineweaver-Burk plot (Fig. S9). In the process, the generated H2O2
was measured by a H2O2 assay kit. The result shows that the
generated H2O2 is increased in response to the elevated
65concentrations of glucose (Fig. 3A), because glucose oxidation could
happen in the present of GOD. A kinetic study of the tandem
catalytic reaction showed that the catalytic activity significantly
increased with the increasing of L-Arg concentrations (Fig. S10) and
the Km of L-Arg is 8.0 mM (Fig. S11). These results also suggest that
70the GOD@Co-FeMOF could catalyze L-Arg into NO in the presence
of glucose, with the reaction orders of 0-1 with respect to either L-
Arg or glucose.
DAF-FM DA probe at different conditions, and the results obtained
25were shown in Fig. 2C and 2D. With the time increasing, GOD@Co-
FeMOF (Fig.2C, curve a), the mixture of GOD@Co-FeMOF and
glucose (curve b) and the mixture of GOD@Co-FeMOF and L-Arg
Fig. 3 The generated (A) H2O2 concentration and (B) NO concentrations
75arising from the reaction between GOD@Co-FeMOF and different
concentrations of glucose.
Fig. 2 (A) FL of DAF-FM DA with Co-FeMOF (a), H2O2 (b), L-Arg (c), glucose (d),
30FeTCPP (e), GOD@Co-FeMOF (f), H2O2 + L-Arg (g), FeTCPP + H2O2 + L-Arg (h),
GOD@Co-FeMOF + H2O2 + L-Arg (i) and Co-FeMOF + H2O2 + L-Arg (j). (B)
Kinetic curves plotting the time-dependent fluorescence intensity at 515 nm
for DAF-FM DA mixing with FeTCPP (a), Co-FeMOF (b) GOD@Co-FeMOF (c)
at the present of H2O2 and L-Arg. Kinetic curves plotting the time-dependent
35fluorescence emission intensity at 515 nm for DAF-FM DA mixing with (C)
GOD@Co-FeMOF (a), GOD@Co-FeMOF + glucose (b), GOD@Co-FeMOF + L-
Arg (c) and (D) GOD + glucose + L-Arg (a), H2O2 + L-Arg (b) and GOD@Co-
FeMOF + glucose + L-Arg (c). 5.0 mM H2O2, 20 mM L-Arg, 1.0 mg/mL glucose,
0.25 mg/mL FeTCPP, and 1.0 mg/mL GOD@Co-FeMOF. Ex=485 nm, Em=515
40nm.
To study the generation of NO concentrations in the tandem
catalytic reaction, the generated NO concentrations in solutions
were measured using a typical Griess assay. Although the rate of
80the L-Arg-H2O2 reaction is slow in neutral solution,19a the generated
NO increases with the increase of time and L-Arg concentration,
and reaches the plateau with 20 mM L-Arg at 30 min in this catalytic
system (Fig. S12). The reason for this phenomenon is that the
generated gluconic acid could accelerate oxidization of L-Arg by
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H2O2,19a in addition, the Co-FeMOF as the catalyst could increase
the rate of reaction. As the concentration of glucose increases, the
amount of generation NO increases (Fig. 3B). For the reaction, the
generated NO amount depends on the produced H2O2 amount, and
the latter relies on the added glucose concentration.
4
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The stability of GOD@Co-FeMOF was measured by FL, XRD and
SEM after five cycles in 0.1 M pH 6.9 and 7.4 PBS (Fig. S13-S17),
which shows that the GOD@Co-FeMOF complex can maintain good
and stable activity, and exhibits excellent catalysis.
6
7
10
In order to demonstrate that GOD@Co-FeMOF can function as
effective catalysts for the generation of NO with endogenous
components. The serum samples obtained from rabbit were
properly diluted by 0.1 M PBS, and then mixed with the DAF-FM DA,
GOD@Co-FeMOF and L-Arg. The result shows that NO is generated
8
9
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clearly indicating the approach could be used in complex sample to
produce NO, and has the potential to use in biomedical field.
In summary, this work designs a simple, biocompatible and
integrated tandem catalyst system based on conjugating GOD on
20Co-FeMOF. Co-FeMOF not only exhibits peroxidase-like activity but
also could be as a support for the GOD immobilization. Besides,
GOD@Co-FeMOF can drive a reaction cascade to allow for in situ
generation of NO via the oxidation of L-Arginine in physiological pH.
This process can thus allow the sustained generation of NO in the
25presence of glucose and L-Arginine, offering a potential solution to
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We gratefully acknowledge National Natural Science Foundation
of China (21705004, 21675084), Natural Science Foundation of
Anhui Province (1808085QB38) and Open Foundation of State Key
Lab of Analytical Chemistry for Life Science, Nanjing University
(SKLACLS1803).
95
100
40Conflicts of interest
There are no conflicts to declare.
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ChemComm
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DOI: 10.1039/C8CC05068F
Graphical Abstract:
Enzyme-immobilized metal-organic framework nanosheets as tandem
catalysts for generation of nitric oxide
By Pinghua Ling,a* Caihua qian,a Feng Gaoa and Jianping Leib*
An enzyme-immobilized metal-organic framework nanosystem was developed as a tandem
catalyst for in-situ generation of nitric oxide in serum samples.