Cu2+-Modified Metal–Organic Framework Nanoparticles: A Peroxidase-Mimicking Nanoenzyme

Article abstract of DOI:10.1002/smll.201703149

The synthesis and characterization of UiO-type metal–organic framework nanoparticles (NMOFs) composed of Zr⁴⁺ ions bridged by 2,2′-bipyridine-5,5′-dicarboxylic acid ligands and the postmodification of the NMOFs with Cu²⁺ ions are described. The resulting Cu²⁺-modified NMOFs, Cu²⁺-NMOFs, exhibit peroxidase-like catalytic activities reflected by the catalyzed oxidation of Amplex-Red to the fluorescent Resorufin by H₂O₂, the catalyzed oxidation of dopamine to aminochrome by H₂O₂, and the catalyzed generation of chemiluminescence in the presence of luminol/H₂O₂. Also, the Cu²⁺-NMOFs mimic NADH peroxidase functions and catalyze the oxidation of dihydronicotinamide adenine dinucleotide, NADH, to nicotinamide adenine dinucleotide, NAD⁺, in the presence of H₂O₂. The Cu²⁺-NMOFs-catalyzed generation of chemiluminescence in the presence of luminol/H₂O₂ is used to develop a glucose sensor by monitoring the H₂O₂ formed by the aerobic oxidation of glucose to gluconic acid in the presence of glucose oxidase. Furthermore, loading the Cu²⁺-NMOFs with fluorescein and activating the catalyzed generation of chemiluminescence in the presence of luminol/H₂O₂ yield an efficient chemiluminescence resonance energy transfer (CRET) process to the fluorescein reflected by the activation of the fluorescence of the dye (λ = 520 nm, CRET efficiency 35%).

Full text of DOI:10.1002/smll.201703149

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Cu2+-Modified Metal–Organic Framework Nanoparticles:
A Peroxidase-Mimicking Nanoenzyme
Wei-Hai Chen, Margarita Vázquez-González, Anna Kozell, Alessandro Cecconello,
and Itamar Willner*
formation of catalytic sites.[15] Many dif-
The synthesis and characterization of UiO-type metal–organic framework
nanoparticles (NMOFs) composed of Zr4+ ions bridged by 2,2′-bipyridine-
5,5′-dicarboxylic acid ligands and the postmodification of the NMOFs with
Cu2+ ions are described. The resulting Cu2+-modified NMOFs, Cu2+-NMOFs,
exhibit peroxidase-like catalytic activities reflected by the catalyzed oxidation
of Amplex-Red to the fluorescent Resorufin by H2O2, the catalyzed oxida-
tion of dopamine to aminochrome by H2O2, and the catalyzed generation of
chemiluminescence in the presence of luminol/H2O2. Also, the Cu2+-NMOFs
mimic NADH peroxidase functions and catalyze the oxidation of dihydronico-
tinamide adenine dinucleotide, NADH, to nicotinamide adenine dinucleotide,
NAD+, in the presence of H2O2. The Cu2+-NMOFs-catalyzed generation of
chemiluminescence in the presence of luminol/H2O2 is used to develop a
glucose sensor by monitoring the H2O2 formed by the aerobic oxidation of
glucose to gluconic acid in the presence of glucose oxidase. Furthermore,
loading the Cu2+-NMOFs with fluorescein and activating the catalyzed
generation of chemiluminescence in the presence of luminol/H2O2 yield
an efficient chemiluminescence resonance energy transfer (CRET) process
to the fluorescein reflected by the activation of the fluorescence of the dye
(λ = 520 nm, CRET efficiency 35%).
ferent chemical transformations were
driven by catalytic MOFs, such as Friedel–
Crafts alkylation reaction,[16] hydrogena-
tion,[17] and oxidation.[18]
Recent research efforts are directed
to the use of nanomaterials or inorganic
nanoparticles as peroxidase-mimicking
catalysts. Hemin/G-quadruplex struc-
tures,[19] metal nanoclusters, or nanoparti-
cles, such as Au,[20] Cu,[21] Pt,[22] or metal
oxide nanoparticles,[23] e.g., Fe2O3 or TiO2
and carbon-based nanomaterials such
as Cu2+-modified carbon dots,[24] carbon
nitride,[24] and graphene oxide[25] reveal
peroxidase-like functions. Also, hemin
embedded in MOFs,[13] and metal-ion-
functionalized MOFs revealed, in analogy
to horseradish peroxidase (HRP), the gen-
eration of chemiluminescence[26] by the
catalyzed oxidation of luminol by H2O2.
In the present study, we report on the
synthesis of UiO-type MOF nanoparti-
cles[27] (NMOFs) composed of Zr4+ ions
bridged by 2,2′-bipyridine-5,5′-dicarboxylic
Metal–organic frameworks (MOFs) represent a broad class
of porous materials composed of metal ions cross-linked by
organic ligands.[1] Besides the interesting structural features of
MOFs,[2] many different applications of MOFs were reported[3]
including their use as drug carriers,[4] catalysts,[5] sensors,[6]
gas storage,[7] separation,[8] optical devices,[9] photocatalysts,[10]
and as micromotors.[11] The use of the highly porous MOFs as
structural scaffolds for the development of catalysts is particu-
larly interesting.[12] Different methods to synthesize MOF-based
catalysts were developed and these included the incorporation
of catalytic complexes into the pores of the MOFs,[13] the incor-
poration of metal complexes as functional ligands of the MOF
frameworks,[14] and the construction of MOFs that include
ligands for the postsynthetic anchoring of metal ions and the
acid ligands, (1), followed by the postmodification of the bipyri-
dine ligands with Cu2+ ions. The Cu2+-modified NMOFs (Cu2+-
NMOFs) reveal peroxidase-mimicking activities. In contrast to
previous studies[28] reporting peroxidase-mimicking functions
of MOFs containing metal ions such as Cu2+ and Fe3+, which
are part of the frameworks, the present catalytic NMOFs include
a metal-ion ligand (bipyridine) as a part of the framework, and
the complexation of metal ions, e.g., Cu2+ to the ligand, yield
the catalytic peroxidase-mimicking NMOFs. The peroxidase-
mimicking functions of the Cu2+-NMOFs are reflected by the
catalyzed oxidation of Amplex-Red by H2O2 to form Resorufin,
the catalyzed H2O2-driven oxidation of dopamine to amino-
chrome, and the catalyzed generation of chemiluminescence
in the presence of luminol/H2O2. The Cu2+-NMOFs-catalyzed
generation of chemiluminescence in the presence of luminol/
H2O2 was further used to develop a chemiluminescence glucose
sensor based on the aerobic oxidation of glucose in the presence
of glucose oxidase. Also, the Cu2+-NMOFs catalyze the oxidation
of dihydronicotinamide adenine dinucleotide, NADH, by H2O2
to form nicotinamide adenine dinucleotide, NAD+, as a NADH
peroxidase-mimicking “nanoenzyme” is described. Finally,
by the incorporation of the fluorescein dye into the pores of
the NMOFs the chemiluminescence generated by the NMOFs
induces a highly efficient chemiluminescence resonance energy
Dr. W.-H. Chen, Dr. M. Vázquez-González, A. Kozell, Dr. A. Cecconello,
Prof. I. Willner
Institute of Chemistry
Center for Nanoscience and Nanotechnology
The Hebrew University of Jerusalem
Jerusalem 91904, Israel
E-mail: willnea@vms.huji.ac.il
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/smll.201703149.
DOI: 10.1002/smll.201703149
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Figure 1. A) Synthesis of the Cu2+-functionalized Zr4+-5,5′-bipyridine carboxylate-bridged metal–organic framework nanoparticles (NMOFs). B) TEM
image of the NMOFs. C) SEM image of the NMOFs.
transfer (CRET) process resulting in the fluorescence of fluores-
cein, λ = 520 nm. The unprecedented efficient CRET process is
attributed to the porous structure of the NMOFs that allows the
concentration of the energy acceptor dye in close proximity to
the chemiluminescence generating sites.
The NMOFs revealed a bipyramidal structure, ≈200 nm
(Figure 1). The powder X-ray diffraction spectra of the NMOFs
before and after modification with Cu2+ ions are identical
(Figure S1, Supporting Information) implying that the struc-
ture of the NMOFs remains intact after modification with the
Cu2+ ions. The surface area and pore sizes of the Cu2+-modified
NMOFs correspond to 246 m2 g−1 and 1.65 nm, respectively
(surface area and pore size of the NMOFs prior to the modi-
fication with Cu2+ 738 m2 g−1 and 1.99 nm, respectively). The
resulting Cu2+-NMOFs were also characterized by X-ray photo-
electron spectroscopy (see Figure S2, Supporting Information,
and the accompanying discussion) and Fourier transform
infrared spectra (see Figure S3, Supporting Information). The
content of Cu2+ ions in the Cu2+-NMOFs was determined by
inductively coupled plasma atomic emission spectroscopy
analysis and it corresponds to 5.5 wt% in the Cu2+-NMOFs.
The Cu2+-NMOFs mimic the function of HRP and cata-
lyze the oxidation of Amplex-Red to the fluorescent Resorufin
by H2O2 (Figure 2A). The fluorescence spectra of Resorufin
Figure 2. A) Schematic Cu2+-NMOFs-catalyzed oxidation of Amplex-Red to the fluorescent Resorufin product by H2O2. B) Fluorescence spectra of the
Resorufin product generated in the catalyzed oxidation of Amplex-Red, 0.2 × 10−3 m, by Cu2+-NMOFs, 20 µg mL−1, using different concentrations of
H2O2: (a) 0 m, (b) 0.05 × 10−3 m, (c) 0.1 × 10−3 m, (d) 0.2 × 10−3 m, (e) 0.5 × 10−3 m, (f) 1 × 10−3 m, (g) 2 × 10−3 m. Inset: derived calibration curve cor-
responding to the fluorescence intensities of Resorufin generated by variable concentrations of H2O2.
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Figure 3. A) Schematic Cu2+-NMOFs-catalyzed oxidation of dopamine by H2O2 to form aminochrome. B) Time-dependent absorbance changes upon
the Cu2+-NMOFs-catalyzed oxidation of different concentrations of dopamine: (a) 0 m, (b) 0.1 × 10−3 m, (c) 0.5 × 10−3 m, (d) 1 × 10−3 m, (e) 2.5 × 10−3 m,
(f) 5 × 10−3 m, (g) 7.5 × 10−3 m, (h) 10 × 10−3 m. In all experiments H2O2 10 × 10−3 m and Cu2+-NMOFs 20 µg mL−1 were used. C) Rates of the oxidation
of dopamine to aminochrome as a function of dopamine concentration by the Cu2+-NMOFs, and the respective control systems: (a) Cu2+-NMOFs
20 µg mL−1, (b) Cu2+ ions and the bipyridine ligand, each concentration is 17.3 × 10−6 m (at similar molar ratio to those of Cu2+/bipyridine ligand
present in the NMOFs). (c) Cu2+ ions only, 17.3 × 10−6 m. In all experiments H2O2 10 × 10−3 m was used. D) Absorbance spectra of the Cu2+-NMOFs
generated aminochrome by the catalyzed oxidation of different concentrations of dopamine for a fixed time interval of 30 min using Cu2+-NMOFs
20 µg mL−1, H2O2 10 × 10−3 m: (a) 0 × 10−3 m, (b) 1 × 10−3 m, (c) 2.5 × 10−3 m, (d) 5 × 10−3 m, (e) 7.5 × 10−3 m, (f) 10 × 10−3 m. Inset: derived calibration
curve corresponding to the absorbance of the resulting aminochrome as a function of the concentrations of dopamine.
generated upon oxidation of Amplex-Red in the presence of dif-
ferent concentrations of H2O2 and Cu2+-NMOFs, 20 µg mL−1,
for a fixed time interval of 30 min are depicted in Figure 2B and
the derived calibration curve is shown in the inset of Figure 2B.
Similarly to HRP, the Cu2+-NMOFs catalyze the H2O2-driven
oxidation of dopamine (2) to aminochrome (3) (Figure 3A). The
time-dependent absorbance changes upon formation of ami-
nochrome in the presence of different concentrations of dopa-
mine, (2), are shown in Figure 3B. As the concentrations of
dopamine increase, the formation of aminochrome is enhanced.
Control experiments revealed that the H2O2-stimulated cata-
lyzed oxidation of dopamine in the presence of only Cu2+ ions
or in the presence of Cu2+ ions and the bipyridine ligand (at
similar molar ratio to those of Cu2+/bipyridine ligand present
in the NMOFs) lead to very inefficient oxidation of dopamine by
H2O2 (Figure 3C, curves (a), (b) and (c), respectively). Presum-
ably, the porous structure of the NMOFs or cooperative effects
of adjacent Cu2+-bpy complexes associated with the NMOFs lead
to the concentration of dopamine at the catalytic sites. Further
support that dopamine is concentrated in the porous Cu2+-
NMOFs structure was obtained by complementary Brunauer–
Emmett–Teller (BET) measurements. While the Cu2+-NMOFs
exhibited a surface area of 246 m2 g−1 and average pore sizes
of 1.65 nm, the Cu2+-NMOFs treated with dopamine revealed
a surface area corresponding to 150 m2 g−1 and average pore
sizes of 1.31 nm. The lower surface area and decreased pore
size of the Cu2+-NMOFs treated with dopamine are consistent
with the occupation of the pores with dopamine. The effective
incorporation of dopamine into the NMOFs is further supported
1
by H-NMR experiments (Figure S4, Supporting Information).
The Cu2+-NMOFs were treated with dopamine, 10 × 10−3 m,
1
and the resulting NMOFs were separated. The H-NMR spec-
trum of the resulting NMOFs is shown in Figure S4 (Supporting
Information). By the integration of the protons associated with
dopamine and the NMOFs ligands we conclude that the molar
ratio of dopamine:NMOFs ligand is 5:1. (For further discussion
of the molar ratio of dopamine to NMOFs, see Figure S4, Sup-
porting Information.) The oxidation of dopamine is controlled
by the concentration of the Cu2+-NMOFs catalyst, and as the
concentration of the NMOFs increases the oxidation of dopa-
mine is faster (Figure S5, Supporting Information). The absorb-
ance spectra of the aminochrome, (3), generated upon oxidation
of different concentrations of dopamine by H2O2, 10 × 10−3 m, in
the presence of 20 µg mL−1 of the Cu2+-NMOFs for a fixed time
interval of 60 min are depicted in Figure 3D, and the derived
calibration curve is displayed in the inset of Figure 3D. In a fur-
ther control experiment, the Cu2+-NMOFs were treated with the
reaction solution for a time interval of 5 h and, subsequently,
the Cu2+-NMOFs were precipitated and separated. The resulting
solution did not show any catalytic activity toward oxidation of
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Figure 4. A) Chemiluminescence spectra generated by the Cu2+-NMOFs-catalyzed oxidation of different concentrations of luminol by H2O2: (a) 0 m,
(b) 0.01 × 10−3 m, (c) 0.025 × 10−3 m, (d) 0.05 × 10−3 m, (e) 0.075 × 10−3 m, (f) 0.1 × 10−3 m, (g) 0.25 × 10−3 m, (h) 0.5 × 10−3 m. In all experiments Cu2+-
NMOFs 100 µg mL−1 and H2O2 2 × 10−3 m were used. B) Chemiluminescence spectra generated by the Cu2+-NMOFs-catalyzed oxidation of luminol,
0.5 × 10−3 m, using different concentrations of H2O2: (a) 0.1 × 10−3 m, (b) 0.25 × 10−3 m, (c) 0.5 × 10−3 m, (d) 1 × 10−3 m, (e) 2 × 10−3 m, (f) 5 × 10−3 m. In
all experiments Cu2+-NMOFs 100 µg mL−1 were used. Inset: derived calibration curve. C) Chemiluminescence spectra generated by the Cu2+-NMOFs
in the presence of luminol/H2O2 and control systems: (a) Cu2+-NMOFs 100 µg mL−1, (b) Cu2+ ions and the bipyridine ligand, each concentration is
86.6 × 10−6 m, (c) Cu2+ ions only, 86.6 × 10−6 m, (d) NMOFs without Cu2+. In all experiments luminol 0.5 × 10−3 m and H2O2 5 × 10−3 m were used. D)
Sensing of glucose via the Cu2+-NMOFs-catalyzed oxidation of luminol generated by the aerobic GOx-catalyzed oxidation of different concentrations of
glucose. Chemiluminescence spectra were recorded after a fixed time interval of 30 min at which the GOx-catalyzed oxidation of glucose was allowed
to generate H2O2. The concentrations of glucose corresponded to: (a) 1 × 10−3 m, (b) 5 × 10−3 m, (c) 10 × 10−3 m, (d) 25 × 10−3 m, (e) 50 × 10−3 m. In all
experiments Cu2+-NMOFs 100 µg mL−1, GOx 4 U mL−1, and luminol 0.5 × 10−3 m were used. Inset: derived calibration curve.
dopamine, upon addition of dopamine/H2O2, implying that no
leakage of Cu2+-ligand complex or degradation of the NMOFs
occurred within this time interval. Furthermore, the Cu2+-
NMOFs used to catalyze the oxidation of dopamine by H2O2
could be separated, and reused for the secondary oxidation of
dopamine by H2O2 with no noticeable decrease in the catalytic
activity of the Cu2+-NMOFs. In fact, such repeated recycling of
the Cu2+-NMOFs was performed for five cycles with no loss in
the catalytic functions of the nanoparticles.
concentrations existing in the NMOFs yield only trace intensi-
ties of chemiluminescence. Presumably, the concentration of
the luminol in the porous NMOFs, in close spatial proximity to
the Cu2+-bipyridine MOF units, leads to the effective chemilumi-
nescence. The successful quantitative assay of H2O2 by the Cu2+-
NMOFs was then applied to develop a glucose sensor. The aerobic
oxidation of glucose, in the presence of glucose oxidase, GOx,
yields gluconic acid and H2O2. As the concentration of H2O2
relates to the concentration of glucose, the sequestered detec-
tion of the biocatalytically generated H2O2 by the Cu2+-NMOFs
stimulated generation of chemiluminescence, in the presence of
luminol, provides a readout signal for the quantitative detection of
glucose. Figure 4D shows the chemiluminescence spectra gener-
ated in the presence of different concentrations of glucose. Figure
4D (inset) shows the resulting calibration curve.
As stated, recent studies reported on diverse nanoparticles
that mimic peroxidase functions. It should be noted, however,
that a fair comparison between the catalytic peroxidase-mim-
icking activities of the different nanoparticles is difficult due
to the following reasons: (i) the different nanoparticles do not
mimic all peroxidase functions, while some of them mimic
several peroxidase functions other peroxidase functions are
lacking. (ii) Quantitative information addressing the concen-
trations of the catalytic sites in the bulk weight supports is
missing. To address the catalytic functions of the Cu2+-NMOFs
The Cu2+-NMOFs also catalyze the oxidation of luminol
by H2O2 resulting in the generation of chemiluminescence
(Figure S6, Supporting Information). Figure 4A shows the chemi-
luminescence spectra generated in the presence of different con-
centrations of luminol (fixed concentrations of H2O2, 2 × 10−3 m,
and Cu2+-NMOFs, 100 µg mL−1), and Figure 4B depicts the
chemiluminescence spectra in the presence of different concen-
trations of H2O2 (fixed concentrations of luminol, 0.5 × 10−3 m,
and Cu2+-NMOFs, 100 µg mL−1). As the concentration of H2O2
increases, the chemiluminescence spectra are intensified. The
resulting calibration curve is presented in Figure 4B (inset). For
the dependence of the chemiluminescence spectra on the concen-
tration of the Cu2+-NMOFs see Figure S7 (Supporting Informa-
tion). Control experiments reveal that the Cu2+-NMOFs are essen-
tial to generate the chemiluminescence (Figure 4C). Cu2+ ions
alone or Cu2+ ions and the bipyridine ligand at the same molar
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Table 1. Comparison of the catalytic performance of different Cu2+-ion-
functionalized nanoparticles toward the catalyzed oxidation of dopamine
to aminochrome by H2O2.
of the Cu2+-C-dots is ≈3-fold higher as compared to the Cu2+
content in the Cu2+-NMOFs, and the catalytic performance of
the Cu2+-C-dots is ≈2.3-fold higher as compared to the Cu2+-
NMOFs. The advantages of the Cu2+-NMOFs are reflected, how-
ever, by the many chemical transformations that mimic native
peroxidases by these catalytic nanoparticles, vide infra.
max [mg NPs mL−1
solution]
Content of Cu2+
ions [wt%]
System
Ref.
V
Cu2+-graphene oxide NPs
Cu2+-C3N4 NPs
0.3
0.24
0.7
21
4.6
14
[25]
[24]
[24]
[33]
The Cu2+-NMOFs, also, mimic the functions of NADH-
peroxidase. In this system, the Cu2+-NMOFs-catalyzed oxidation
of NADH to NAD+ by H2O2 proceeds (Figure 5A). Figure 5B
shows the time-dependent oxidation of different concentrations
of NADH (probed by following the decrease in the absorbance
Cu2+-C-dots
Cu2+-Fe2+
lates
/
3+-cyanometa-
0.34
17.8
of NADH at λ = 340 nm) in the presence of H2O2, 2 × 10−3
m
Cu2+-NMOFs
0.095
5.5
This study
and Cu2+-NMOFs, 20 µg mL−1. As the concentration of NADH
increases, the rate of oxidation of NADH is enhanced (Figure 5B,
inset). The rate of NADH oxidation is, also, controlled by the
concentration of the Cu2+-NMOFs (see Figure S8, Supporting
Information) and by the concentration of H2O2. Figure 5C
shows the rate of oxidation of NADH by different concentra-
tions of H2O2 (fixed concentrations of NADH, 0.5 × 10−3 m, and
of Cu2+-NMOFs, 20 µg mL−1). For the resulting calibration curve
showing the rate of NADH oxidation as a function of H2O2 con-
centration, see Figure S9 (Supporting Information). An impor-
tant issue that needs to be addressed relates to the proof that
NADH is oxidized to the biologically active cofactor, NAD+,
rather than to the biologically inactive cofactor dimer (NAD)2. To
as compared to other Cu2+-ion-functionalized nanocatalysts, we
selected several Cu2+-ion catalytic systems that identified the
content of the catalytic sites, and compared the systems toward
the identical peroxidase-mimicking transformation, e.g., the
catalyzed oxidation of dopamine to aminochrome. The results
are summarized in Table 1. One may realize that the Cu2+-
NMOFs exhibit lower catalytic performance toward the oxida-
tion of dopamine to aminochrome by H2O2. For example, the
Cu2+ ions loading on the Cu2+-C3N4 is very similar to the Cu2+
loading on the Cu2+-NMOFs. The catalytic activity of the latter
catalyst is, however, ≈2-fold lower. Similarly, the Cu2+ loading
Figure 5. A) Schematic Cu2+-NMOFs-catalyzed oxidation of NADH to NAD+ by H2O2 and the application of the catalytic process as a NAD+-regen-
eration process for biocatalytic transformations. B) Time-dependent absorbance changes upon the Cu2+-NMOFs (20 µg mL−1) catalyzed oxidation of
different concentrations of NADH by H2O2 2 × 10−3 m. NADH concentrations correspond to: (a) 0 m, (b) 0.005 × 10−3 m, (c) 0.01 × 10−3 m, (d) 0.025 ×
10−3 m, (e) 0.05 × 10−3 m, (f) 0.1 × 10−3 m, (g) 0.25 × 10−3 m. Inset: calibration curve corresponding to the rate of NADH oxidation as a function of NADH
concentration. C) Time-dependent absorbance changes upon the Cu2+-NMOFs-catalyzed oxidation of NADH, 0.5 × 10−3 m, by different concentrations
of H2O2: (a) 0 m, (b) 0.1 × 10−3 m, (c) 0.2 × 10−3 m, (d) 0.5 × 10−3 m, (e) 1 × 10−3 m, (f) 2 × 10−3 m, (g) 5 × 10−3 m, (h) 10 × 10−3 m. D) Time-dependent
absorbance changes upon the Cu2+-NMOFs-catalyzed oxidation of NADH by H2O2 to form NAD+ and the subsequent regeneration of NADH by the
AlcDH-catalyzed oxidation of ethanol to acetaldehyde. The initial solid curve represents the time-dependent Cu2+-NMOFs-catalyzed oxidation of NADH
by H2O2 (Cu2+-NMOFs 20 µg mL−1, NADH 0.1 × 10−3 m, and H2O2 2 × 10−3 m). At the point marked with an arrow ethanol 30 × 10−3 m and AlcDH
50 U were added to the system, resulting in the recovery of NADH.
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confirm the formation of the NAD+ cofactor, the Cu2+-NMOFs-
catalyzed oxidation of NADH by H2O2 was activated for 60
min to allow the depletion of the NADH absorbance, Figure
5D. Afterward, ethanol and the enzyme alcohol dehydroge-
nase, AlcDH, were added to the system. The spectral features
of NADH were recovered by ≈95% and the absorbance of the
recovered NADH retained a steady-state value. The recovery of
the NADH absorbance implies that the biologically active NAD+
cofactor was formed as a result of the NAD+-dependent AlcDH-
catalyzed oxidation of ethanol to acetaldehyde. Furthermore, we
note that after completion of the oxidation of NADH to NAD+
by the H2O2, the Cu2+-NMOFs were precipitated, and the sepa-
rated Cu2+-NMOFs were reapplied for the catalyzed oxidation of
NADH. The Cu2+-NMOFs catalytic function was unaffected and
the oxidation of NADH followed the results shown in Figure 5.
These experiments were repeated three times and only a minute
effect on the activities of the Cu2+-NMOFs was observed (≈10%
decrease in the activity due to the loss of the Cu2+-NMOFs in
the separation steps). These results imply that the Cu2+-NMOFs
provide an effective catalyst for the oxidation of NADH to NAD+.
Note that control experiments revealed that Cu2+ ions or Cu2+
ions in the presence of the bipyridine ligand (at similar molar
concentrations existing in the NMOFs) did not show any NADH
peroxidase activities under identical conditions used with the
Cu2+-NMOFs, thus demonstrating the unique catalytic proper-
ties of Cu2+-NMOFs as NADH peroxidase-mimicking nano-
enzymes. The effective Cu2+-NMOFs-catalyzed oxidation of
NADH by H2O2 is attributed to the concentration of NADH in
the porous structure of the NMOFs, in close proximity to the
catalytic sites. This is supported by complementary surface
area analysis. While the Cu2+-NMOFs revealed a surface area
of 246 m2 g−1 and average pore size of 1.65 nm, the NMOFs
treated with NADH exhibited a surface area corresponding to
142 m2 g−1 and pore size of 1.26 nm, respectively. The lower
surface area and decrease in the pore size of the NMOFs, after
their treatment with NADH, imply that the pores are loaded
with the NADH substrate. Beyond the demonstration that the
Cu2+-NMOFs act as an NADH-peroxidase-mimicking catalyst,
the system introduces a novel mean to regenerate the NAD+
cofactor, thus allowing the application of the NMOFs as catalyst
to drive biotechnological processes driven by NAD+-dependent
enzymes.
Finally, we applied the Cu2+-NMOFs as a functional mate-
rial for stimulating a CRET process. Previous studies indi-
cated that the chemiluminescence generated by HRP using the
luminol/H2O2 system can induce, albeit at very low efficiency,
a CRET process to organic dyes, e.g., fluorescein.[29] Also, the
hemin/G-quadruplex HRP-mimicking DNAzyme linked to
semiconductor quantum dots[30] or associated in supramolec-
ular complexes with organic dyes[31] leads, in the presence of
luminol/H2O2, to inefficient CRET processes that result in the
luminescence of the quantum dots or the fluorescence of the
organic dyes, without external excitation of the chromophores.
The porous structure of the Cu2+-NMOFs suggested that the
incorporation of an acceptor dye in the porous material in close
proximity to the catalytic chemiluminescence Cu2+-NMOFs
sites could stimulate the CRET process. Accordingly, the Cu2+-
NMOFs were loaded with the fluorescein dye (Figure 6A). The
Figure 6. A) Incorporation of fluorescein to Cu2+-NMOFs. B) The stimulation of the chemiluminescence resonance energy transfer (CRET) from the
Cu2+-NMOFs to fluorescein in the presence of luminol/H2O2. The CRET process leads to the fluorescence of fluorescein at λ = 520 nm. C) Lumines-
cence spectra comprising the luminol/H2O2 chemiluminescence band (I) and the fluorescence band of fluorescein generated by the CRET process
(II) in the presence of different concentrations of the NMOFs: (a) 10 µg mL−1, (b) 20 µg mL−1, (c) 50 µg mL−1, (d) 75 µg mL−1, (e) 100 µg mL−1. In all
experiments luminol 0.5 × 10−3 m and H2O2 5 × 10−3 m were used.
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dye-loaded Cu2+-NMOFs were subjected to the luminol/H2O2
chemiluminescence generation system (Figure 6B). Figure 6C
depicts the luminescence spectrum of the system. Interestingly,
besides the chemiluminescence spectrum of the luminol/H2O2
system, at 425 nm, an intense fluorescence band of fluorescein
at λ = 520 nm is observed. The fluorescence of fluorescein is
originated from a CRET process in the Cu2+-NMOFs. Control
experiments revealed that no CRET-induced fluorescence of flu-
orescein is observed in the presence of Cu2+ ions or Cu2+/bipy-
ridine ligand assemblies. From the intensity of the absorbance
band of fluorescein, we estimated[32] the CRET efficiency to
be 35%. This unprecedented CRET efficiency is attributed to
the concentration of the acceptor dye in the porous matrix of
the NMOFs in close spatial proximity to the dense catalytic
sites of Cu2+-NMOFs. These CRET generating NMOFs could be
applied as optical reporter for sensors, e.g., metal ions (Cu2+) or
biosensors, e.g., probing H2O2 generated by oxidases.
Keywords
chemiluminescence resonance energy transfer (CRET), dopamine,
glucose, NADH, sensors
Received: September 18, 2017
Revised: October 15, 2017
Published online:
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Conflict of Interest
The authors declare no conflict of interest.
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