The phosphatase-like activity of zirconium oxide nanoparticles and their application in near-infrared intracellular imaging

Article abstract of DOI:10.1039/d0tb00450b

In this study, the phosphatase mimetic activity of zirconium oxide nanoparticles (ZrO₂NPs) has been demonstrated. They can effectively catalyze the dephosphorylation of a series of commercial fluorogenic and chromogenic substrates of natural phosphatases. Compared with natural phosphatases, ZrO₂NPs possess several advantages such as low cost, facile preparation procedures, and high stability in a broader pH range or at high temperatures. In addition, the activity of ZrO₂NPs toward some important biomolecules was investigated. The ZrO₂NPs can catalyze the dephosphorylation of ATP ando-phospho-l-tyrosine, but they cannot react with DNA strands. These data are important for the further bio-related applications of ZrO₂NPs. Finally, the potential application of ZrO₂NPs in intracellular imaging is also demonstrated by using a near-infrared fluorescent substrate of alkaline phosphatase.

Full text of DOI:10.1039/d0tb00450b

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ISSN 2050-750X
PAPER
Wei Wei, Guanghui Ma et al.
Macrophage responses to the physical burden of cell-sized
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DOI: 10.1039/D0TB00450B
ARTICLE
The phosphatase-like activity of zirconium oxide nanoparticle and
its application in near-infrared intracellular imaging
Xilu Hu,a Ting Huang,b Hong Liao,a Lianzhe Hu*a and Min Wang*b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
In this study, the phosphatase mimetic activity of zirconium oxide nanoparticles (ZrO2 NPs) is demonstrated. It can
effectivtly catalyze the dephosphorylation of a series of commercial fluorogenic and chromogenic substrates of natural
phosphatase. Compared with natural phosphatase, ZrO2 NPs possess several advantages such as low-cost, easy to
preparation, and high stability in a broader pH range or high temperature. In addition, the activity of ZrO2 NPs toward
some important biomolecules was investigated. The ZrO2 NPs can catalyze the dephosphorylation of ATP and o-phospho-L-
tyrosine, but it can not react with DNA strands. These data are important for the further bio-related applications of ZrO2
NPs. Finally, the potential application of ZrO2 NPs in intracellular imaging is also demonstrated by using a near-infrared
fluorescent
substrate
of
alkaline
phosphatase.
nanozyme is significant due to the wide applications of natural
phosphatase. In enzyme linked immunoassays, horseradish
peroxidase (HRP) and ALP are the most commonly used
enzyme labels. A variety of substrates of HRP and ALP are
commercially available, which facilitates the investigation of
the enzymatic properties of their artificial mimics. For
instance, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)
(ABTS), 3,3',5,5'-tetramethylbenzidine (TMB), and Amplex red
are frequently used in the studies of peroxidase nanozymes.22
However, compared with peroxidase nanozymes, the
investigations on phosphatase nanozyme are very rare.23
Recently, the phosphatase-like activity of CeO2 NPs are
reported.24-27 Most of the studies are focus on the
chromogenic substrate p-nitrophenyl phosphate (p-NPP),
which has limited their bio-analytical applications such as
imaging.28
In this study, the phosphatase-like activity of ZrO2 NPs is
demonstrated. Although ZrO2 based materials have been
widely used in the field of phosphopeptide enrichment due to
the high affinity between the Zr(IV) and phosphate, the
catalytic activity of ZrO2 NPs has been long-termly
neglected.29,30 As shown in scheme 1, we found ZrO2 NPs can
effectively catalyze the dephosphorylation of fluorogenic
substrate 4-methylumbelliferyl phosphate (4-MUP) to produce
the fluorescent molecule 4-methylumbelliferon (4-MU). To
further demonstrate the phosphatase-like activity, a series of
phosphatase substrates were studied in the presence of ZrO2
NPs. More importantly, the dephosphorylation of typical
biological molecules were studied, including ATP, o-phospho-L-
tyrosine, and DNA strands. Among these biological molecules,
o-phospho-L-tyrosine was investigated due to its importance in
the protein phosphorylation. The catalytic activity of ZrO2 NPs
toward these biologically relevant molecules has important
implications for their further bio-related applications.
Introduction
Nanomaterials with enzyme-like characteristics (also called
nanozymes) have received increasing attention because of
their possibility as alternatives to natural enzymes.1-3 As a new
kind of artificial enzymes, nanozymes exhibit intrinsic
advantages such as high stability, easy to large-scale
preparation, and low cost.4-6 Nanozymes have been
demonstrated as useful materials in various fields including
biomedicine, molecular sensing, and environmental
protection.7-11 However, most of the developed naozymes are
oxidoreductase mimics, such as peroxidase mimics, oxidase
mimics, catalase mimics, and superoxide dismutase mimics.12-
16
In comparison, natural enzymes are known to catalyze a
large variety of different types of reactions including redox
reaction, hydrolytic reaction, ligation reaction, decomposition
reaction, etc. It is interesting to develop new nanozymes that
can catalyze other types of reaction beyond redox reactions.
For example, Zelikin et al. reported the identification of
apparent pan-glycosidase activity in nanozymes and its
application in prodrug therapies.17
Alkaline phosphatase (ALP) is an important hydrolytic
enzyme in the phosphate metabolism.18 It plays essential roles
in regulating cellular functions and disease pathogenesis. It is
also used as a biocatalyst in commercial enzyme linked
immunoassays.19-21 Therefore, the exploration of phosphatase
a. Chongqing Key Laboratory of Green Synthesis and Applications, College of
Chemistry, Chongqing Normal University, Chongqing 401331, China. E-mail:
lianzhehu@cqnu.edu.cn
b. School of Pharmaceutical Sciences, Chongqing University, Chongqing 401331,
China. E-mail: wang_min@cqu.edu.cn
Electronic Supplementary Information (ESI) available: DLS data, XPS spectra,
kinetic studies, fluorescent spectra, gel electrophoresis, absorbance spectra, and
cytotoxic experiment. See DOI: 10.1039/x0xx00000x
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µg/mL ZrO2 NPs, the dephosphorylation reaction of 4-MUP
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was greatly accelerated. Strong fluoreDscOeIn: 1c0e.10w39a/sD0oTbBs0e0r4v5e0dB
from the mixture, which is attributed to the formation of
fluorescent product 4-MU. The dephosphorylation of 4-MUP
was further investigated in the different concentrations of
ZrO2 NPs. As shown in Fig. S4, the reaction rate was increased
with the increasing concentrations of ZrO2 NPs. As a further
control, we checked whether the dephosphorylation was due
to the ZrO2 NPs or from the dissolved Zr(VI) ions. To
demonstrate this, the ZrO2 NPs sample was centrifuged and
the supernatant was tested. As shown in Fig. S5, the
supernatant of ZrO2 NPs was catalytically inactive, which
suggests the phosphatase-like activity was originated from the
ZrO2 NPs. Interestingly, we found that Zr(OH)4 and Zr(VI) could
also catalyze the dephosphorylation of 4-MUP, although their
activity were lower than ZrO2 NPs (Fig. S5). From these data,
Zr(VI) plays the key role in the phosphatase-like catalytic
activity. p-NPP can strongly bind with ZrO2 NPs due to the high
affinity of Zr(IV) with phosphate group. The high Lewis acidity
of Zr(IV) can facilitate the hydrolysis of the phosphate ester by
hydroxide ion, which may result in the phosphatase-like
activity of ZrO2 NPs.
Scheme 1. Schematic illustration of the dephosphorylation reaction of 4-MUP catalyzed
by ZrO2 NPs.
Results and discussion
Characterization of the ZrO2 NPs
Fig. 1A shows the TEM image of the ZrO2 NPs used in our
experiments. The individual ZrO2 NP has a size range from 20
nm to 40 nm, and slight aggregation of the particles can be
observed from the TEM images. The TEM images with higher
resolution were also provided in Fig S1. Fig. 1B shows the
dynamic light scattering spectra of the ZrO2 NPs, the
hydrodynamic diameters of ZrO2 NPs were in the range from
90 nm to 200 nm, which confirms the slight aggregation. The
zeta potential of ZrO2 NPs was also measured, an average zeta
potential value of -13.4 mV was obtained (Fig. S2). The XPS
analysis was also performed to investigate the composition
and chemical state of the ZrO2 NPs (Fig. S3). The characteristic
peaks corresponding to Zr 4p (29.98 eV), Zr 3d (182.56 eV), Zr
3p (332.50 and 345.93 eV) and O 1s (529.91 eV) are clearly
identified in the survey spectrum. As shown in Fig. S1B, the
Zr4+ 3d peak was observed and can be decomposed into four
peaks. The peaks located at 182.11 and 184.50 eV were
associated to Zr-O bonds while the peaks located at 181.67
and 184.11 eV were attributed to metallic Zr bonds(Zr−Zr).31,32
These results are agreed well with the components of ZrO2 NPs.
Fig. 2 The time-dependent fluorescence changes of 20 mM HEPES buffer (pH 8.5)
(curve a), 5 µM 4-MUP (curve b), and 5 µM 4-MUP in the presence of 100 µg/mL ZrO2
NPs (curve c) under excitation at 360 nm. Inset: the corresponding images of the
solutions under 365 nm UV irradiation.
Fig. 3A shows the fluorescent spectra of 5 µM 4-MUP in the
presence of a few common metal oxide NPs. After incubation
of 4-MUP with these metal oxide NPs for 30 min, only the ZrO2
NPs/4-MUP system showed obvious fluorescence emission.
This means only ZrO2 NPs showed apparent phosphatase-like
activity in our experiment. The steady-state kinetics of the
phosphatase-like activity of ZrO2 NPs was then investigated. As
shown in Fig. S6, typical Michaelis-Menten curves can be
obtained for ZrO2 NPs with 4-MUP as the substrate. The
comparison of the kinetic parameters of ZrO2 NPs with that of
CeO2 NPs and ALP was shown in Table S1. The apparent Km
Fig.
1 (A) The TEM image of the ZrO2 NPs, the scale bar is 500 nm. (B) The
hydrodynamic size distribution of the ZrO2 NPs.
The phosphatase-like activity ZrO2 NPs
The phosphatase-like activity of ZrO2 NPs was then studied
using 4-MUP as the representative substrate. As shown in Fig.
2, the dephosphorylation rate of 5 µM 4-MUP was very slow in
20 mM pH 8.5 HEPES buffer, and negligible fluorescence signal
was observed without the catalyst. In the presence of 100
2 | J. Name., 2012, 00, 1-3
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value of ZrO2 NPs is lower than that of CeO2 NPs, suggesting robustness of ZrO2 NPs makes them suitable for a wider scope
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DOI: 10.1039/D0TB00450B
the ZrO2 NPs have higher affinity to 4-MUP as compared with of application in biochemistry and industry, especially the
CeO2 NPs. Not surprisingly, the affinity of ALP with 4-MUP is dephosphorylation reaction in industry at high temperatures.
much stronger than that of ZrO2 NPs and CeO2 NPs, which is Recently, a various of Ce-based materials have been reported
due to the specific binding pocket in the protein structure.
to exhibit phosphatase-like activity.24-27 In comparison, the Zr-
based phosphatase mimic is low cost (more suitable for large-
scale use) as Zr element is more abundant in the earth.
Fig. 3 (A) The fluorescent spectra of 5 µM 4-MUP in the presence of 20 mM HEPES
buffer (pH 8.5) (a), MgO NPs (b), CuO NPs (c), Fe3O4 NPs (d), TiO2 NPs (e), ZnO NPs (f),
Al2O3 (g), SiO2 (h), SnO2 (i), NiO (j), Co3O4 (k), and ZrO2 NPs (l). (B) The corresponding
images of the solutions under 365 nm UV irradiation. All the concentrations of the NPs
are 100 µg/mL, and the 4-MUP was incubated with the NPs for 30 min before the
measurement.
Fig. 4 Comparison of the stability of ZrO2 NPs and ALP. (A) ZrO2 NPs and ALP were first
incubated at a range of values of pH from 2.5 to 10 for 2 h and then their phosphatase
activity were measured under standard condition. (B) ZrO2 NPs and ALP were first
incubated at a range of temperatures between 15 to 90 °C for 2 h and then their
phosphatase activity were measured under standard condition.
The advantages of ZrO2 NPs as a phosphatase mimic
Although the catalytic efficiency of ZrO2 NPs is lower than that
of natural ALP, ZrO2 NPs has unique advantages as a
phosphatase nanozyme. First, the natural ALP is expensive due
to the difficulties in preparation and purification, while ZrO2
NPs is cost effective and easy to preparation in the lab. Second,
as inorganic nanomaterials, ZrO2 NPs are more stable than
natural enzymes. To demonstrate this, ZrO2 NPs and ALP were
firstly incubated at pH 2.5-10 or at 15-90 °C for 2 h, and then
their activities were measured at standard conditions (room
temperature, pH 8.5). Fig. 4 shows that ALP loses activity
significantly after incubation at low pH or high temperatures.
In comparison, ZrO2 NPs retained high catalytic activity after
the incubation at a wide range of pH and temperature. The
The activity ZrO2 NPs toward different substrates
The catalytic activities of ZrO2 NPs toward other substrates of
ALP were investigated. p-NPP is
a
commonly used
chromogenic substrate of ALP in enzyme linked immunoassays.
As shown in Fig. S7, the ZrO2 NPs can also catalyze the
dephosphorylation of p-NPP to generate yellow color product,
which indicates the formation of p-nitrophenol. The result
suggests the potential of ZrO2 NPs as ALP alternative in
commercial enzyme linked immunoassays.
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The activities of ZrO2 NPs toward some biological relevant
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DOI: 10.1039/D0TB00450B
molecules were measured. The amount of free phosphate
produced in the reaction of ZrO2 NPs with ATP, o-phospho-L-
tyrosine, and a random DNA sequence was monitored using a
commercial malachite green assay kit, respectively. Fig. S8
shows that ZrO2 NPs can react with ATP and o-phospho-L-
tyrosine to generate free phosphate ions, although the
catalytic hydrolysis of ATP and o-phospho-L-tyrosine did not
occur as readily as with p-NPP. We also found that ZrO2 NPs
can not react with DNA, as no free phosphate ion was
monitored after incubated ZrO2 NPs with DNA strands. Unlike
p-NPP with one ester oxygen atom, the phosphate groups in
DNA strands have two ester linkers. The phosphorus atom is
too sterically hindered to undergo the nucleophilic substitution,
which might be the reason of the low activity of ZrO2 NPs with
DNA. Recently, Liu et al. reported CeO2 NPs can cleave single-
stranded DNA oligonucleotides, and the cleavage efficiency
was increased with the temperature.33 As shown in Fig. S9,
ZrO2 NPs can not cleave DNA strand either at room
temperature or at 60 °C. All these data are important for the
further use of ZrO2 NPs in biological applications.
Fig. 5 Confocal images of HeLa cells (top) and ZrO2 NPs loaded HeLa cells (bottom) after
staining with Mito-Tracker Green and DiI. 10 µM of SunRed phosphate was exposed to
both the HeLa cells and nanoceria-loaded HeLa cells before imaging. Scale bars are 20
μm.
Conclusions
In conclusion, the phosphatase mimetic activity of ZrO2 NPs
was studied using different substrates of natural phosphatase.
Compared with natural ALP, the ZrO2 NPs shows several
advantages such as low cost, easy to preparation, and high
stability under harsh conditions. In addition, the activities of
ZrO2 NPs toward the biological relevant molecules were
important for the further bio-related applications of ZrO2 NPs.
Compared with Ce-based phosphatase mimics, the Zr-based
phosphatase mimic is more suitable for large-scale use
because Zr element is more abundant in the earth. Overall,
this study will not only promote the studies on hydrolytic
nanozymes, but also stimulate the applications of ZrO2 NPs as
a phosphatase mimic in various of areas such as enzyme linked
immunoassays, biosensors, and bio-imaging.
Application in fluorescent imaging
The phosphatase mimetic activity of ZrO2 NPs was then used
for intracellular imaging. Because ALP is widely used in enzyme
linked immunoassays, various of fluorogenic substrates of ALP
with are commercially available. The phosphatase mimetic
activity of ZrO2 NPs was further demonstrated by using the
green emission substrate (fluorescein diphosphate) and the
near-infrared emission substrate (SunRed phosphate). As
shown in Fig. S10, ZrO2 NPs can catalyze the
dephosphorylation of both fluorescein diphosphate and
SunRed phosphate to generate the corresponding fluorescent
product. The near-infrared substrate SunRed phosphate was
selected for the intracellular imaging due to the low
background of near-infrared imaging. Before the intracellular
imaging, the cytotoxicity of ZrO2 NPs was evaluated. As shown
in Fig. S11, the cell viability was greater than 90% over a wide
range concentration of ZrO2 NPs, indicating the good
biocompatibility of the ZrO2 NPs.
Fig. 5 shows the results of the intracellular imaging by using
SunRed phosphate as the substrate. The HeLa cells and the
ZrO2 NPs-loaded HeLa cells were stained by mitochondrial-
selective dye Mito-Tracker Green and membrane dye DiI,
respectively. As shown in Fig. 5, only ZrO2 NPs loaded cells
showed the near-infrared emission, which was due to the
hydrolysis of SunRed phosphate catalyzed by ZrO2 NPs. In
contrast, the untreated cells showed no near-infrared emission.
The data implies that ZrO2 NPs have great potential in
intracellular fluorescent imaging. It should be noted, the bio-
imaging using nanozyme has its unique advantages. First, it can
be used for tracking the nanoparticles loaded into cells,
especially the nanoparticles that can not generate
fluorescence by themselves. Second, if some substances inside
the cell could enhance or inhibit the catalytic activity of
nanozyme, the method could be used for imaging the
corresponding substance indirectly.
Experimental Section
Reagents and materials
Alkaline phosphatase (ALP), 4-methylumbelliferyl phosphate
(4-MUP), 4-methylumbelliferon (4-MU), malachite green
phosphate assay kit, ATP, o-phospho-L-tyrosine, and p-
nitrophenyl phosphate (p-NPP) were purchased from Sigma.
Fluorescein diphosphate (FDP) and SunRed phosphate were
obtained from AAT Bioquest, Inc (USA). Mito-Tracker Green
and DiIC18(3) (DiI) were supplied by Beyotime Biotechnology
Co., Ltd. The synthetic oligonucleotides were purchased from
Sangon Biotech Co., Ltd (Shanghai, China). Their sequences are
as follows:
S1: 5'-TCTTTGGCATACTTGATCACCAGGCACTTGTACTG-3' (S1);
S2: 5'-FAM-TCTTTGGCATACTTGATCACCAGGCACTTGTACTG-3'
ZrO2 nanoparticles (NPs) (20-40 nm), TiO2 NPs (20-40 nm),
SiO2 NPs (20 nm), ZnO NPs (20 nm), Al2O3 NPs (150-500 nm),
SnO2 NPs (10-30 nm), MgO NPs (20 nm), and Fe3O4 NPs (<10
nm) were purchased from XFNano Materials Tech Co., Ltd
(Nanjing China). CuO (<50 nm) NPs was obtained from Sigma.
Co3O4 NPs (5 nm) was synthesized according to Zheng's work (J.
4 | J. Name., 2012, 00, 1-3
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Mater. Chem. B, 2018, 6, 6858). CeO2 NPs (5 nm) was prepared
ARTICLE
The cleavage of DNA by CeO2 NPs and ZrOV2iewNAPrtisclewOnelirnee
according to Perez's work (Angew. Chem. Int. Ed. 2009, 48, investigated as follows. First, 25 µM oDf ODI:N10A.10s3tr9a/Dn0dTBS0204w50aBs
2308-2312).
reacted with 100 µg/mL ZrO2 NPs or 100 µg/mL CeO2 NPs for 1
h at different temperature ( 25 °C and 60 °C, respectively) .
Then, the reaction products were analyzed by using denatured
Apparatus
Transmission electron microscopy (TEM) images were gel electrophoresis. As the DNA strand S2 was labeled with
obtained on a Tecnai G2 20 S-TWIN transmission electron fluorophore FAM, the gel was imaged without further staining.
microscope. Dynamic light scattering (DLS) measurements
Cell culture, cell viabilities and confocal imaging
were performed on a Zetasizer Nano ZS (Malvern Instruments,
UK). XPS measurement was carried out on an X-ray
HeLa cells were cultured in DMEM containing 10% FBS and 1%
photoelectron spectrometer using monochromatized Al Kα penicillin-streptomycin at 37 °C with 5% CO2. To evaluate the
radiation (Thermo Fisher Scientific, USA). Confocal images cytotoxicity of ZrO2 NPs, the cells were seeded onto a 96-well
were acquired using a Leica TCS SP8 laser scanning confocal plate at a density of 5.0×104 cells per well. After culture for 24
microscope (Leica Microsystems, Germany). Fluorescence h, the cells were treated with ZrO2 NPs at different
measurements were carried out on a PerkinElmer Model LS-55 concentrations (i.e., 0, 5, 10, 20, 50 and 80 μg/mL) for 24 h and
Luminescence Spectrometer. Absorption spectra were washed with PBS. Subsequently, 100 μL of MTT solution (0.5
measured using
(Shimadzu, Japan).
a
UV2550 UV-Vis Spectrophotometer mg/mL in PBS) was introduced into each well, followed by
incubation for 4 h under dark environment and removal of the
supernatant. Finally, 100 μL of dimethyl sulfoxide was added
into each well to dissolve the formazan crystals before
Kinetic analysis and relative activity comparison
The phosphatase-like catalytic property of ZrO2 NPs was acquiring the absorbance at 490 nm on an iMark Microplate
investigated using steady-state kinetics. First, the fluorescent Reader (Bio-Rad, USA).
data were carried out by recording the fluorescent value at
For confocal imaging, HeLa cells were seeded on glass-
455 nm in scanning kinetics mode at room temperature. Then, bottom dishes (35 mm Petri dish) and cultured for 24 h. The
the fluorescence values were back-calculated to the cells were then treated without and with 50 μg/mL of ZrO2 NPs
concentration of 4-MU, based on the fact that the fluorescent for 2 h and washed with PBS. For staining the mitochondria,
intensity was proportional to the concentrations of the the cells were incubated with Mito-Tracker Green (100 nM in
o
reaction product 4-MU. Finally, the reaction rate was obtained normal cell culture medium) for 15 min at 37 C and washed
according to the speed of 4-MU formation. Experiments were with PBS. For staining the cell membrane, the cells were
o
conducted using 100 µg/mL ZrO2 NPs in 20 mM HEPES buffer incubated with DiI solution (5 μM in PBS) for 10 min at 37 C
(pH 8.5) containing different concentrations of 4-MUP, and the and then washed with HEPES buffer(10 mM, pH 7.4). Finally,
fluorescent intensities were recorded at a reaction time of 2 the cells were imaged in the presence of HEPES buffer
minutes. The Michaelis-Menten constant was calculated using containing 250 nM near-IR probe SunRed phosphate.
Lineweaver-Burk plots of the double reciprocal of the
Michaelis-Menten equation.
To investigate the influence of solution pH on the ZrO2 NPs
Conflicts of interest
and ALP activity, ZrO2 NPs and ALP were firstly incubated at
different pH values (from pH 2.5 to 10) for 2 h, then their
activities were measured at under standard condition (pH 8.5,
room temperature). To test the thermo-stability of ZrO2 NPs
and ALP, ZrO2 NPs and ALP were firstly incubated in 20 mM
HEPES buffer (pH 8.5) at given temperatures for 2 h, and then
their activities were measured at room temperature.
There are no conflicts to declare.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (NSFC, 21605012 and 21705012), the
Science and Technology Research Program of Chongqing
Municipal Education Commission (KJQN201900522), and the
Young Scholar of Chongqing Bayu Scholars (BYQNCS2019006).
Catalytic activity of ZrO2 NPs toward biomolecules
The dephosphorylation of ATP, o-phospho-L-tyrosine, and
DNA by ZrO2 NPs were investigated by monitoring the amount
of inorganic phosphate that liberated into the solution. First,
25 µM of biomolecule (ATP, o-phospho-L-tyrosine, or DNA
strand S1) was mixed with 100 µg/mL ZrO2 NPs for 30 minutes
at room temperature. Then, the amount of inorganic
phosphate in the mixture was evaluated using a commercial
malachite green assay kit. The malachite green solution was
prepared according to the kit instructions, and the absorbance
values at 620 nm represents the existence of free phosphate in
the solutions.
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Journal of Materials Chemistry B
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DOI: 10.1039/D0TB00450B
TOC:
The phosphatase-like activity of ZrO2 nanoparticles is reported, which has found
applications in intracellular imaging.