Ascorbate Oxidase Mimetic Activity of Copper(II) Oxide Nanoparticles

Article abstract of DOI:10.1002/cbic.201900595

Although oxidase mimetic nanozymes have been widely investigated, specific biological molecules have rarely been explored as substrates, particularly in the case of ascorbate oxidase (AAO) mimetic nanozymes. Herein, we demonstrate for the first time that copper(II) oxide nanoparticles (CuO NPs) catalyze the oxidation of ascorbic acid (AA) by dissolved O₂ (as a green oxidant) to form dehydroascorbic acid (DHAA), thus functioning as a new kind of AAO mimic. Under neutral conditions, the Michaelis–Menten constant of CuO NPs (0.1302 mm) is similar to that of AAO (0.0840 mm). Furthermore, the robustness of CuO NPs is greater than that of AAO, thus making them suitable for applications under various conditions. As a demonstration, a fluorescence AA sensor based on the AAO mimetic activity of CuO NPs was developed. To obtain a fluorescent product, o-phenylenediamine (OPDA) was used to react with the DHAA produced by the oxidation of AA catalyzed by CuO NPs. The developed sensor was cost-effective and easy to fabricate and exhibited high selectivity/sensitivity with a wide linear range (1.25×10^?6 to 1.125×10^?4 m) and a low detection limit (3.2×10^?8 m). The results are expected to aid in expanding the applicability of oxidase mimetic nanozymes in a variety of fields such as biology, medicine, and detection science.

Full text of DOI:10.1002/cbic.201900595

Accepted Article
Title: Ascorbate oxidase mimetic activity of cupric oxide nanoparticles
Authors: Shao-Bin He, Ai-Ling Hu, Quan-Quan Zhuang, Hua-Ping
Peng, Hao-Hua Deng, Wei Chen, and Guo-Lin Hong
This manuscript has been accepted after peer review and appears as an
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To be cited as: ChemBioChem 10.1002/cbic.201900595
Link to VoR: http://dx.doi.org/10.1002/cbic.201900595
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10.1002/cbic.201900595
ChemBioChem
ARTICLE
Ascorbate oxidase mimetic activity of cupric oxide nanoparticles
Shao-Bin He,#[a] Ai-Ling Hu,#[a,b] Quan-Quan Zhuang,[a] Hua-Ping Peng,[a] Hao-Hua Deng,*[a] Wei
Chen,*[a] and Guo-Lin Hong*[c]
Abstract: Although oxidase mimetic nanozymes have been widely
investigated, specific biological molecules have rarely been explored
as substrates, especially for ascorbate oxidase (AAO) mimetic
nanozymes. Herein, for the first time, we demonstrate that cupric
oxide nanoparticles (CuO NPs) exhibit as a catalyst toward the
oxidation of ascorbic acid (AA) by dissolved O2 as a green oxidant to
form dehydroascorbic acid (DHAA), thus revealing a new kind of
AAO mimic. Under neutral conditions, Michaelis–Menten constant of
CuO NPs (0.1302 mM) is similar to that of AAO (0.0840 mM).
Furthermore, the robustness of CuO NPs is greater than that of AAO
rendering them suitable for applications under various conditions. As
a demonstration, a fluorescence AA sensor was established based
on AAO mimetic activity of CuO NPs. To obtain a fluorescent
product, o-phenylenediamine (OPDA) was used to react with DHAA
produced by CuO NP-catalyzed oxidation of AA. The fabricated
sensor was cost-effective, easy to fabricate, and exhibited high
selectivity/sensitivity with a wide linear range (1.25 × 10-6 to 1.125 ×
10-4 M) and a low limit of detection (3.2 × 10-8 M). The results are
expected to aid in expanding the applicability of oxidase mimetic
nanozymes in a variety of fields such as biology, medicine, and
detection science.
nanomaterials have excellent oxidase mimetic activity, their
widespread application has been prohibited by poor substrate
selectivity. Up to now, most substrates for oxidase mimetic
nanozymes have been non-specific model substrates (e.g.,
3,3',5,5'-tetramethylbenzidine, o-phenylenediamine (OPDA), 2,
2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)).[4] However,
few studies have examined specific biological molecules as
substrates.[5] Hence, the exploration of nanozymes that catalyze
specific biomolecular substrates is highly desirable.
Cu-based materials have been studied extensively owing to its
special properties and extensive applications in cross-coupling
catalysis, immunoassays, high-temperature superconductor,
antimicrobial applications, biosensors, and field-emission
emitters.[6] Furthermore, our earlier work revealed water-soluble
cupric oxide nanoparticles (CuO NPs) to have intrinsic enzyme
mimetic activity, igniting widespread interest in the application of
these promising nanozymes to replace enzymes.[7] Notably, self-
cascade reactions catalyzed by CuO NPs with specific biological
molecules (e.g. cysteine and glutathione) allowed oxidase and
peroxidase mimetic activities to be established for the efficient
detection of cysteine, glutathione, and Ag+, providing a new
design concept for the use of CuO NPs.[8] These studies suggest
that further exploration of the oxidase-like activities of CuO NPs
toward specific biological molecules would broaden their
applications and provide insight into the nanozyme-based
catalytic mechanism.
Ascorbate oxidase (AAO), a multi-copper enzyme, catalyzes the
reduction of O2 into water with synchronized oxidation of
ascorbic acid (AA).[9] Mertz and co-workers reported an apparent
correlation between the enzyme activity of AAO and cell
enlargement in maize root cells, with the enzyme activity of AAO
mainly associated with the cell wall fraction.[10] Furthermore, it
has been suggested that AAO could be a important role in
dynamic system for oxygen management in plants and regulate
the growth of plant cells by working in accordance with AA.[11]
However, the potential applicability of AAO in the fields of
biomedicine, biosensor, and biotechnology is not yet fully
understood. Similar to some other natural enzymes, AAO has
some serious disadvantages such as a low thermal stability, a
high cost, and tedious preparation/purification processes.[12] It's
should be noted that few studies have investigated the
ascorbate oxidase mimetic activity of nanomaterials.[3d,13] Thus
efforts are required to obtain enzyme mimetics for AAO.
Introduction
Over the last decade, various nanozymes (nanomaterials with
intrinsic enzyme mimetic activities) have been found that
successfully mimic natural enzymes.[1] Among them, peroxidase
nanozyme, which uses H2O2 as electron acceptor, has attracted
the most attention due to its wide application. However, in some
applications, the addition of H2O2 is undesirable owing to its
instability and destructive nature. Therefore, it is desirable to
develop oxidase mimics that can catalyze oxidation reactions
using O2 as green oxidant. Asati and co-workers showed for the
first time that CeO2 nanoparticles (NPs) possess oxidase
mimetic activity.[2] Subsequent efforts have explored other
nanozymes with oxidase activity, including Au, Ag, Pt, Au@Pt,
Au@Ag, and Au@Pd/Pt.[3] Although these types of
[a]
Prof. W. Chen, Dr. S.B. He, A.L. Hu, Dr. Q.Q. Zhuang, Prof. H.P. Peng,
Prof. H.H. Deng
Department of Pharmaceutical Analysis
Fujian Medical University
Herein, we found that CuO NPs possess AAO mimetic activity,
catalyzing the oxidation of AA to dehydroascorbic acid (DHAA)
in the presence of O2, and the good AAO mimetic activity and
stability of CuO NPs makes them suitable for various
applications. To demonstration the applicability of CuO NPs, a
fluorescence sensor for AA detection was established based on
CuO NP-catalyzed AA oxidation and DHAA-induced formation of
fluorescent 3-(1, 2-dihydroxyethyl) furo[3,4-b]quinoxaline-1-one
(DFQ) from non-fluorescent OPDA.[14] After optimizing the
detection response, this simple, low-cost, selective, and
sensitive fluorometric method yielded satisfactory results for the
Fuzhou 350004, China
E-mail: chenandhu@163.com, DHH8908@163.com (H.H. Deng)
A.L. Hu
Department of Pharmacy, First Hospital of Qinhuangdao, Qinhuangdao
066000, China
Prof. G.L. Hong
Department of Laboratory Medicine
The First Affiliated Hospital of Xiamen University
Xiamen 361003, China
18860089899@139.com (G.L. Hong)
Corresponding author. Tel./fax: +86 591 22862016.
These authors contributed equally to this work.
[b]
[c]
*
#
Supporting information for this article is given via a link at the end of
the document.
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10.1002/cbic.201900595
ChemBioChem
ARTICLE
quantitative sensing of AA in real samples. These investigations
of AAO mimetic activity of CuO NPs enrich the family of oxidase
mimetic nanozymes and provide the opportunity to expand their
applications in biology, medicine, and detection science.
were performed and chromatographs depicting AA, DHAA, and
AA-O2-CuO NPs are shown in Fig. 2B. Notably, when monitoring
at 265 nm, a strong peak was observed for AA (trace a) but a
poor response was obtained for DHAA (trace b), and the
products of the AA-O2-CuO NPs reaction could not be observed
(trace c) using a C18 column with methanol-KH2PO4 (0.05 M, pH
= 6) as the mobile phase. Monitoring at 210 nm revealed that AA
eluted at 3 min (trace d), whereas DHAA eluted at 3.6 min (trace
e). Meanwhile, the addition of CuO NPs to AA (trace f) resulted
in a remarkable decrease in the peak at 3 min and the
appearance of a peak at 3.6 min, which indicated that AA can be
oxidized to DHAA by O2 in the presence of CuO NPs.
Results and Discussion
CuO NPs were prepared via a previously reported quick-
precipitation method.[7b] The morphology and particle size of the
as-prepared CuO NPs were characterized by TEM (Fig. 1A).
The TEM image in Fig. 1A revealed that CuO NPs consists of
spherical particles with
a uniform morphology. The size
Fig. 2C depicts the time-dependent (0–180 s) changes in the
absorption spectra upon oxidation of AA by O2 in the presence
of CuO NPs. The absorbance band of AA at 265 nm decreases,
completely disappearing after 180 s, demonstrates that CuO
NPs quickly catalyze the oxidation of AA to DHAA. To determine
whether AA interacts with CuO NPs or Cu2+ leached from the
CuO NPs under the examined reaction conditions, Cu2+ was
obtained by incubating CuO NPs in the reaction buffer for 30 min
and then removing CuO NPs from the solution by the use of
centrifugation. As shown in Fig. 2D, changing rate at 265 nm
increases sequential during the reaction process in the presence
of CuO NPs. However, there is not any marked change in the
changing rate at 265 nm when Cu2+ solution was used instead of
the CuO NPs under the same reaction conditions. These results
indicate that the ascorbate oxidase mimetic activity can be
attributed to CuO NPs rather than Cu2+. Hence, it can draw a
conclusion that CuO NPs have AAO mimetic activity that can
catalyze the oxidation of AA to DHAA in the presence of O2.
distribution analysis was adjusted by Gaussian distribution and
the result revealed CuO NPs with an average diameter of
approximately 6.8 nm (Fig. 1B). A typical XRD pattern of CuO
NPs was also indicated in Fig. 1C. It could be noted that the
matrix of these NPs is indexed as a CuO phase with a
monoclinic structure (Lattice constants: a = 4.68 Å, b = 3.42 Å, c
= 5.13 Å, and β = 99.54o, JCPDS No. 72-0629). Moreover, no
obvious impurity peaks were found confirming the high phase
purity of the obtained CuO NPs. The diffraction pattern of CuO
NPs has broadened peaks ascribed to the small particle size
which is in agreement with the result from the TEM image.
Figure 1. (A) TEM image of CuO NPs. (B) Size distribution analysis (100 random
NPs) of CuO NPs. (C) XRD pattern of CuO NPs.
The oxidation of AA as catalyzed by CuO NPs in dissolved O2
was monitored by recording the absorption at 265 nm using
UV/Vis spectroscopy (Fig. 2A). AA exhibits a distinct absorption
maximum at 265 nm (trace a). However, when CuO NPs were
incubated with AA, an obvious decrease of absorbance at 265
nm was noted, demonstrating that AA was consumed in reaction
(trace b). Moreover, experiments under de-oxygenated
conditions (trace c) supported the involvement of O2 in the
reaction because no significant decrease in the absorbance at
265 nm was observed when N2 was bubbled.
Figure 2. (A) UV/Vis spectra of (a) AA, (b) AA-O2-CuO NPs, and (c) AA-CuO NPs
under a N2 atmosphere for 20 min. (B) Chromatogram at 265 nm of (a) AA, (b)
DHAA, and (c) AA-O2-CuO NPs, and chromatograms at 210 nm of (d) AA, (e) DHAA,
and (f) AA-O2-CuO NPs (tR is the abbreviation of retention time). (C) Time-
dependent spectral changes (a, 0 s; b, 30 s; c, 60 s; d, 120 s; e, 180 s) of AA
corresponding to CuO NP-catalyzed oxidation (f, solution containing DHAA instead
of AA). (D) Evolution of the time-dependent absorbance-changing rate at 265 nm for
AA in the presence of (a) CuO NPs and (b) leaching solution (Cu2+).
To obtain insight into the catalytic process of the AA-O2-CuO
NPs reaction, high-performance liquid chromatography analyses
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ARTICLE
To further evaluate the ascorbate oxidase mimetic activity of
CuO NPs, the effects of the concentration of CuO NPs, pH, and
temperature on the AA-O2-CuO NPs system were investigated
by recording the absorption (265 nm) using UV/Vis spectroscopy.
Fig. 3A shows the relationship between the changing rate of AA
at 265 nm and CuO NPs concentration. The changing rate
increases until the concentration of CuO NPs reaches 0.5 mg/L
and then remains approximately constant at higher CuO NP
concentrations. The stability of ascorbyl radicals is known to
vary with reaction conditions such as pH and temperature. In
this case, the optimal pH and temperature were observed to be
approximately 7.0–9.0 (Fig. 3B) and 40–60 °C (Fig. 3C),
respectively. Thus, we employed pH 7.0 and 45 °C as the
optimal conditions in subsequent experiments.
Figure 4. Steady-state kinetic assays with (A) CuO NPs and (B) AAO.
Table 1 Comparison of steady-state kinetic parameters of CuO NPs and AAO.
Catalyst
Km (mM) Vmax (10-4 mM/s) [E] (10-5 mM) Kcat (s-1) a
CuO NPs
AAO
0.1302
0.0840
9.9
6.5
3.67
26.98 b
363.13
0.179
a Kcat = Vmax / [E], where [E] is the concentration of the catalyst.
b See the calculation details in the Supporting Information.
As an inorganic material, CuO NPs are expected to be stable
under harsh environments over a much longer time than AAO,
which tends to lose its catalytic activity after exposure to
extreme pH and high temperatures. To highlight the superiority
of stability of CuO NPs, the catalytic activities of CuO NPs and
AAO were tested after incubation at a range of pH values and
temperatures for 1 h. After treatment at a pH lower than 5 or
higher than 9 (Fig. 5A) or temperatures greater than 40 °C (Fig.
5B), the catalytic activity of AAO decreased obviously. Unlike
AAO, CuO NPs were stable over a wide range of pH values (3–
12, Fig. 5A) and temperatures (10–90 °C, Fig. 5B). Taken
together, the robust stability and considerable ascorbate oxidase
mimetic activity makes CuO NPs suitable for analytical
applications.
Figure 3. Effects of (A) concentration of CuO NPs, (B) pH, and (C) temperature on
the absorbance-changing rate of the AA-O2-CuO NPs system.
To obtain the steady-state kinetic parameters, we further studied
the catalytic behavior of CuO NPs and AAO with AA as
substrate, built on enzyme kinetics theory and methods. The
Michaelis–Menten model is one of the most widely used models
and most commonly used to study enzymatic reaction. In Fig. 4,
the solid circles are the experimental data and the solid curves
are fits to the Michaelis–Menten model. In a range of AA
concentration, Michaelis–Menten equations were achieved for
CuO NPs (Fig. 4A) and AAO (Fig. 4B), and the Michaelis–
Menten constant (Km) can be obtained. The kinetics of the
enzyme reaction can be characterized by the parameters Km and
catalytic rate constants (Kcat).Under a neutral condition, the Km of
CuO NPs (0.1302 mM) is slightly higher than that of AAO
(0.0840 mM). The catalytic performance of CuO NPs could be
attributed to their full surface exposure of CuO atoms. In order to
effectively compare the catalytic behavior of CuO NPs and AAO,
the catalytic rate constants (Kcat) of CuO NPs (26.98 s-1) and
AAO (363.13 s-1) were calculated, respectively. These relatively
minor differences in kinetic parameters demonstrate that CuO
NPs and AAO possess comparable affinities for AA (Table 1).
Figure 5. Relative activities of CuO NPs and AAO after incubation for 1 h at various
(A) pH values and (B) temperatures.
Scheme 1. Schematic illustration of the ascorbate oxidase mimetic activity of CuO
NPs and OPDA as a probe for fluorescence sensing of AA.
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10.1002/cbic.201900595
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For sensing of AA, OPDA can be used as a probe for the final
product (DHAA) of the reaction of the AA-O2-CuO NPs system.
OPDA is non fluorescent, but the specific reaction between
OPDA and DHAA produces DFQ, which is strong fluorescent.
Thus using the AAO mimetic activity of CuO NPs, AA can be
rapidly oxidized by dissolved O2 to DHAA, which then reacts with
OPDA to produce DFQ, thus providing a unique method for AA
sensing (Scheme 1). As is illustrated in Fig. 6, both the OPDA-
O2 (trace a) and OPDA-O2-CuO NPs system (trace b) exhibit
almost no fluorescence intensity. Although AA can undergo
reaction conditions, the fluorescence intensity at 425 nm was
proportional to the AA concentration in the range of 1.25 × 10-6
to 1.125 × 10-4 M with a low limit of detection (LOD) of 3.2 × 10-8
M (Fig. 7B). The RSD was 1.8% at 1.125 × 10-4 M AA (n = 6).
The analytical performance of this OPDA-AA-O2-CuO NPs
sensor was compared with that of typical AA detection methods
reported in recent years (Table 2). Compared with these other
methods, the approach proposed herein shows a wider linear
range, better sensitivity, and relatively low detection limits.
To confirm the selectivity of this method for AA, experiments
were conducted using cysteine, homo-cysteine, GSH, glucose,
lactose, maltose, fructose, citric acid, and uric acid (Fig. 7C). A
high intensity was obtained in the presence of AA, whereas no
obvious signals were observed for the other compounds, even
though their concentrations (1.25 mM) were 10-fold higher than
that of AA (0.125 mM). By combining the CuO NP-catalyzed
oxidation of AA with the selectivity of OPDA for DHAA, the
system achieved high selectivity for AA.
auto-oxidation in the absence of
a catalyst, only weak
fluorescence was observed in OPDA-O2-AA system (trace c),
whereas the fluorescence intensity of the OPDA-AA-O2-CuO
NPs system (trace d) was greatly enhanced, with an emission
maximum at 425 nm when excited at 350 nm. The fluorescence
spectrum of the OPDA-AA-O2-CuO NPs system (trace d) is
similar in shape to that of OPDA-DHAA system (Fig. S1), which
confirms that OPDA reacts directly with DHAA obtained by the
Cu NP-catalyzed oxidation of AA. The inset photograph in
Scheme 1 clearly shows luminescence of the OPDA-AA-O2-CuO
NPs system under a UV lamp.
Figure 6. Fluorescence emission spectra of (a) OPDA, (b) OPDA-CuO NPs, (c)
OPDA-AA, and (d) OPDA-AA-CuO NPs (excitation wavelength = 350 nm).
The OPDA-AA-O2-CuO NPs system was subsequently
optimized to establish the optimum analytical conditions for AA
sensing (Fig. S2). The effect of OPDA concentration was tested
in the range from 6.25 × 10-3 to 0.625 mM and the maximum
fluorescence intensity was observed at 0.25 mM of OPDA,
which could be chosen as the optimum concentration for further
experiments (Fig. S2A). The effect of pH in reaction solution was
examined by adjusting the pH from 3 to 9. As shown in Fig. S2B,
the fluorescence intensity remains approximately constant at pH
from 3–6 and then decreases dramatically from pH 6.5 to pH 9.
Therefore, pH 5.0 was selected as the optimum pH for further
experiments. Fig. S2C shows that the fluorescence intensity
increased significantly as the incubation temperature increased
to 20 °C. Slight variations were observed in the fluorescence
intensity at temperature of 20–30 °C, and then the fluorescence
intensity decreased gradually above 30 °C. Therefore, 30 °C
was selected as the reaction temperature for further experiments.
The kinetics of reaction between DHAA and OPDA to produce a
fluorescence signal was also investigated (Fig. S2D). Initially,
the signal increases, with a maximum fluorescence obtained at
20 min. Hence, reaction time of 20 min was chosen for further
experiments.
Figure 7. (A) Fluorescence emission spectra (excitation wavelength = 350 nm) of
the OPDA-AA-O2-CuO NPs system at different AA concentrations (10-6 M): (a) 0, (b)
1.25, (c) 6.25, (d) 12.5, (e) 37.5, (f) 62.5, (g) 87.5, and (h) 112.5. (B) Calibration plot
constructed using the fluorescence intensity at 425 nm. (C) Fluorescence intensities
(at 425 nm) of OPDA-CuO NPs in the presence of foreign substances: 1, cysteine; 2,
homocysteine; 3, GSH; 4, glucose; 5, lactose; 6, maltose; 7, fructose; 8, citric acid; 9,
uric acid; 10, AA (1-9: 1.25 mM; 10: 0.125 mM).
To explore the applicability and feasibility of the developed
sensing method, it was applied to the determination of AA in real
samples such as commercial Vitamin C tablets and a lemon
beverage. As shown in Table 3, results obtained using the
OPDA-AA-O2-CuO NPs sensor were in good agreement with
those determined using the Standard Chinese Pharmacopoeia
Iodimetric method (standard method). The practical applicability
of the OPDA-AA-O2-CuO NPs biosensor was verified through
standard addition experiments. The RSD was less than 5.0%
and the average recovery of real samples was between 92.6%
and 110.6%. These results indicate that the OPDA-AA-O2-CuO
NPs sensor meets the requirements for microanalysis and is
practical for the determination of AA in commercial tablets and
lemon beverages. Moreover, both the F-test and t-test showed
Fig. 7A depicts the fluorescence response of OPDA-AA-O2-CuO
NPs system in the presence of AA concentration. Under optimal
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10.1002/cbic.201900595
ChemBioChem
ARTICLE
no significant statistical difference at the 95% confidence level
between the proposed and standard analytical methods.
Experimental Section
Cupric acetate, H2O2 (30wt%), AA, glucose, maltose, D-fructose,
L-cysteine, uric acid, citric acid, oxalic acid, ethanol, NaCl, GSH,
lactose, sodium hydroxide, and OPDA were purchased from
Sinopharm Chemical Reagent Co. Ltd. (China). DHAA and AAO
were purchased from Sigma Co. Ltd. (Germany).
Table 2 Comparison of typical methods for AA sensing.
Method
Linear range (M)
2 × 10-6 to 2.06 × 10-4
1.2 × 10−6 to 1.61 × 10−3
5 × 10−5 to 2 × 10-2
6 × 10−6 to 8 × 10−5
2 × 10-6 to 5 × 10-5
1 × 10-7 to 2.5 × 10-6
1 × 10-5 to 1 × 10-3
0 to 5 × 10-5
LOD (M)
9 × 10-7
4.3 × 10-7
1.5 × 10-6
8 × 10-7
1.6 × 10-7
4.9 × 10-8
1.4 × 10-6
4 × 10-8
3.6 × 10-6
2 × 10-7
-
Ref.
15
Cyclic voltammetry
Amperometry
16
Transmission electron microscopy (TEM) images were collected
with a JEM-2100 TEM (JEOL, Japan). X-ray diffraction (XRD)
patterns were recorded with a Bruker D8 Advance diffractometer.
A UV-2450 spectrophotometer (Shimadzu, Japan) was used to
record the UV/Vis absorption spectra. High-performance liquid
chromatography analyses were performed using an HPLC-1200
17
18
19
20
instrument (Agilent Technologies, USA).
A Cary Eclipse
Colorimetry
fluorescence spectrometer (Agilent Technologies, USA) was
used to record the fluorescence spectra.
21
22
5 × 10-6 to 3 × 10-5
1.5 × 10-6 to 1 × 10-5
3 × 10-5 to 1.0 × 10-4
-
23
CuO NPs were prepared via a previously reported quick-
precipitation method.[7b] In order to investigate the ascorbate
oxidase mimetic activity of the CuO NPs, catalytic oxidation of
AA in the presence of O2 was conducted. Typically, an
experiment was carried out at 45 °C by using 0.5 mg/L CuO NPs
in a reaction volume of 4.0 mL (200 mM phosphate buffer, pH =
7.0) with 500 µL of 0.125 mM AA as substrates. Then, the
24
Fluorescence
25
2 × 10-6
3.2 × 10-8
26
OPDA-AA-O2-CuO NPs
1.25 × 10-6 to 1.125 × 10-4
This work
products were monitored at
λ = 265 nm using UV/Vis
spectroscopy. To test the stability, the CuO NPs were incubated
at different temperatures (10–90 °C) or pH values (3–12) for 1 h,
and then their ascorbate oxidase mimetic activities were
measured under the standard conditions mentioned above.
Table 3 Analytical results for AA contents of samples determined using the OPDA-
AA-O2-CuO NPs sensor and the standard method.
Proposed
method
Standard
method
Sample
F-test
2.4
t-test
0.5
First, 500 µL of AA of various concentrations and 50 µL of 40
mg/L CuO NPs were added into 450 µL of phosphate buffer
solution (200 mM, pH 7.0). After incubating at 40 °C for 6 min,
2.5 mL of phosphate buffer solution (200 mM, pH 5.0) and 500
µL of 2 mM OPDA were added, and the resulting mixture was
incubated for another 20 min at 30 °C. The fluorescence spectra
of the resulting reaction solution were recorded at an excitation
wavelength of 350 nm.
5.8 ± 0.18
7.1 ± 0.33
3.08 ±0.12
5.5 ± 0.28
7.2 ± 0.26
3.33 ± 0.15
Vitamin C tablets (mg)
Lemon-beverage (mM)
1.6
0.1
1.6
1.0
F0.05,2,2 = 19.00, t0.05,4 = 2.776, n = 3
For the pharmaceutical analysis of AA, samples of vitamin C
tablets were dissolved and diluted 10-fold in purified water after
centrifugation, and analyzed directly. The lemon beverage
samples were diluted (1:10 v/v) to obtain an AA concentration
within the linear range of response. For AA analysis, results of
the proposed method were compared with those of the Standard
Chinese Pharmacopoeia Iodimetric method.
Conclusions
In conclusion, CuO NPs exhibited as a catalyst toward the
oxidation of AA by O2 as a green oxidant to form DHAA under
mild conditions. The apparent steady-state kinetic parameters
indicated that this new type of ascorbate oxidase mimic and
AAO have similar affinities for AA, whereas CuO NPs catalyze
the oxidation reaction more rapidly than AAO. Moreover, CuO
NPs exhibited considerable stability and almost constant
Acknowledgements
The authors gratefully acknowledge the financial support of the
National Natural Science Foundation of China (21175023), the
Program for Innovative Leading Talents in Fujian Province
(2016B016), and the Program for Innovative Research Team in
Science and Technology in Fujian Province University
(2018B033).
catalytic activity over
a wide range of storage pH and
temperature. By coupling the CuO NP-catalyzed oxidation of AA
to the reaction between OPDA and DHAA, which produces a
fluorescent product, a sensitive/selective assay for AA was
realized with a lowest detection limit of 3.2 × 10-8 M. In addition,
this proposed method was successfully applied to determine AA
in real samples. Owing to the excellent ascorbate oxidase
mimetic activity of CuO NPs, there is considerable scope for
improving their enzyme mimetic activity and exploring their
potential applications in biology, medicine, and detection science.
Keywords: Cupric oxide nanoparticles • ascorbate oxidase
mimetic activity
• ascorbic acid • o-phenylenediamine •
fluorometric sensor
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10.1002/cbic.201900595
ChemBioChem
ARTICLE
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10.1002/cbic.201900595
ChemBioChem
ARTICLE
Entry for the Table of Contents
ARTICLE
Herein, for the first time, we
demonstrate that cupric oxide
nanoparticles (CuO NPs) exhibit as
a catalyst toward the oxidation of
ascorbic acid (AA) by dissolved O2
Shao-Bin He,#[a] Ai-Ling Hu,#[a,b]
Quan-Quan Zhuang,[a] Hua-Ping
Peng,[a] Hao-Hua Deng,*[a] Wei
Chen,*[a] and Guo-Lin Hong*[c]
Page No. – Page No.
as
a
green oxidant to form
dehydroascorbic acid (DHAA), thus
revealing a new kind of ascorbate
oxidase (AAO) mimic. The results
are expected to aid in expanding the
applicability of oxidase mimetic
nanozymes in a variety of fields such
as biology, medicine, and detection
science.
Ascorbate oxidase mimetic activity
of cupric oxide nanoparticles
This article is protected by copyright. All rights reserved.