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Engineering Two-Dimensional Pd Nanoplates with Exposed Highly
Active {100} Facets Toward Colorimetric Acid Phosphatase
Detection
Chuanxia Chen,
†,∥
Wendong Liu,
†,∥
Pengjuan Ni,
†
Yuanyuan Jiang,*
,†
Chenghui Zhang,
†
Bo Wang,
†
Jinkai Li,
†
Bingqiang Cao,
‡
Yizhong Lu,*
,†
and Wei Chen*
,§
†
School of Materials Science and Engineering, University of Jinan, Jinan 250022, China
‡
Department of Physics and Institute of Laser, Qufu Normal University, Qufu 273165, China
§
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences,
Changchun 130022, China
*
SSupporting Information
ABSTRACT: Enzyme-like activity and efficiency of nanoma-
terials are strongly controlled by their size, composition, and
structure, and hence the structural parameters need to be
optimized. Here, we report that two-dimensional Pd nano-
plates enclosed by {100}-facets [{100}PdSP@rGO] exhibit
substantially enhanced intrinsic oxidase-like activities relative
to the {111}-facets ones and Pd nanocubes in catalyzing the
chromogenic reaction of 3,3′,5,5′-tetramethylbenzidine. By
taking ascorbic acid 2-phosphate as the substrate, which
transforms to ascorbic acid in the presence of acid phosphatase (ACP), the {100}PdSP@rGO could be used as an efficient
nanozyme for colorimetric ACP detection without resorting to destructive H2O2. A good linear relationship from 0.01 to 6.0
mU/mL with a detection limit of 8.3 μU/mL is obtained, which is better than most previously reported ACP assays.
Importantly, the excellent assay performance could be successfully applied to ACP determination in serum samples with high
accuracy. This study demonstrates that the enzyme-like activity of nanomaterials could be finely tuned simultaneously by
controlling their exposed crystal facets and high specific surface area.
KEYWORDS: two-dimensional Pd nanoplates, facets, oxidase-like activity, acid phosphatase, colorimetric
■INTRODUCTION
Acid phosphatase (ACP), which catalyzes the dephosphor-
ylation of orthophosphate monoesters under acidic conditions,
is a ubiquitous phosphatase in various mammalian tissues and
fluids. An abnormal elevation of the ACP level in serum
samples may indicate various diseases like prostatic diseases,
kidney disorder, hyperparathyroidism, and multiple myeloma.
1
Thus, ACP can serve as a pivotal serum biomarker and a
prognostic indicator for these diseases. Therefore, construction
of reliable and cost-effective ACP assays with high sensitivity
and selectivity is of great clinical significance.
Through quantifying alcohols generated from ACP-catalyzed
dephosphorylation of various orthophosphoric monoesters,
several methods have been developed to probe ACP activity,
including electrochemistry,
2,3
colorimetry,
4−7
fluorimetry,
8−19
potentiometric immunoassay,
20
and chromatography.
21
Among these methods, colorimetry, featuring simplicity, cost-
effectiveness, rapidity, and visibility, has attracted extensive
attention. Generally, routine colorimetric ACP assays directly
monitor the colored hydrolysis products of specially
synthesized substrates by UV−vis absorption spectroscopy or
even by the naked eye.
5−7
However, they always suffer from
poor sensitivity. Moreover, synthesis of novel organic
substrates is ineluctably complicated and laborious and needs
harmful organic reagents. As a result, developing sensitive
colorimetric ACP activity assays with reliable performance and
simple procedures is still a challenging task.
Profiting from the intrinsic amplification of signals in an
exponential manner and the easily detectable signal output,
enzyme-based colorimetry endows detection with exceptional
ultrasensitivity and operability. Recently, the noble metal-based
nanomaterials as enzyme mimics and detection tools,
particularly Au, Pt, Ru, Ir, and Pd, have received considerable
attention owing to the merits of low cost, good stability against
denaturing and high catalytic activities.
22−24
Among them, Pd-
based nanomaterials have drawn extensive research interest
over the past several years. Recent studies have shown that the
enzyme-mimetic properties of Pd-based nanomaterials can be
efficiently tuned by controlling their composition, size,
structure and crystal facets. For instance, Yin and co-workers
Received: September 16, 2019
Accepted: November 25, 2019
Published: November 25, 2019
Research Article
www.acsami.org
Cite This: ACS Appl. Mater. Interfaces 2019, 11, 47564−47570
© 2019 American Chemical Society 47564 DOI: 10.1021/acsami.9b16279
ACS Appl. Mater. Interfaces 2019, 11, 47564−47570
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reported that {111}-faceted Pd octahedrons with lower surface
energy have greater intrinsic antioxidant enzyme-like activity
than {100}-faceted Pd nanocubes with higher surface energy.
25
Zhou demonstrated that concave Pd nanocrystals enclosed by
high-index facets could catalyze ascorbate autoxidation more
efficiently than the nanocrystals with low-index facets.
26
However, although extensive progress has been achieved for
Pd-based enzyme-like catalysts, few reports focus on the
enzyme mimic study and application of their two-dimensional
(2D) nanostructures.
27
Oxidase mimics can be attractive alternatives for designing
sensors. They can catalyze the oxidation of organic substrates
without the need for unstable and destructive H2O2. Detection
of target can be easily achieved via modulating the oxidase-like
activity of nanozymes. 2-Phospho-L-ascorbic acid (AAP) is a
typical phosphatase substrate widely adopted in various
phosphatase activity assays. Enzymatic hydrolysis of AAP by
ACP yields ascorbic acid (AA) and the orthophosphate ion.
Due to its glorious reducibility, AA is capable of scavenging
radicals and reducing certain targets. That means AA can
obstruct the oxidase-like activity of nanozymes by reacting with
the generated radicals and colored products.
In this work, we discover an intriguing phenomenon that
palladium square nanoplates on reduced graphene oxide
(PdSP@rGO) exhibit a facet-dependent oxidase-like activity.
Pd nanoplates enclosed by {100}-facets [{100}PdSP@rGO] is
more active than the {111}-facets ones. Moreover, the 2D
PdSP@rGO shows enhanced activity compared to the Pd
nanocubes. When AA is introduced, the {100}PdSP@rGO-
catalyzed chromogenic reaction of 3,3′,5,5′-tetramethylbenzi-
dine (TMB) is hindered, together with fading in color and a
decrease in absorbance. Colorimetric detection of ACP activity
is thus rationally achieved using AAP as a substrate and the
chromogenic reaction of TMB as a signal amplifier. It is worth
noting that both the ACP-triggered hydrolysis of AAP and the
{100}PdSP@rGO-catalyzed oxidation of TMB work well in
acidic medium and at physiological temperature. Therefore,
one-step detection of ACP can be anticipated reasonably
through a facile mix-and-readout manner. This makes the
analysis simpler and quicker.
■EXPERIMENTAL SECTION
Chemicals and Materials. GO was purchased from XFNANO at
Nanjing. Na2PdCl4, poly(vinyl pyrrolidone) (PVP; MW = 55,000),
KBr, KCl, AA, AAP, ACP, o-phenylenediamine (OPD), TMB,
tryptophan, catalase (CAT), trypsin, glucose oxidase (GOx), bull
serum albumin (BSA), and alkaline phosphatase (ALP) were obtained
from Sigma-Aldrich (USA). Lysozyme, pancreatin, and pepsin were
bought from Shanghai Macklin Biochemical Technology Co., Ltd.
(China). Nitrotetrazolium blue chloride (NBT) and 2,2′-azinobis(3-
ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS) were
obtained from Aladdin Industrial Corporation (Shanghai, China).
Synthesis of Carbonyl Group-Modified GO. Typically, 50 mg
of GO was placed in a tube furnace maintained at 60 °C for 1 h under
an Ar atmosphere. When it cooled to room temperature, the solid was
obtained and used without any post-treatment.
Synthesis of {100}PdSP@rGO. In a typical synthesis,
28
KBr (120
mg), AA (12 mg), and PVP (50 mg) were dissolved into 8 mL of
water, which contains 12.96 mg of GO in a glass pressure vessel. The
mixture was preheated at 80 °C under stirring for 10 min. Then, 12
mg of Na2PdCl4in 3 mL of water was quickly added. The vessel was
then capped and kept at 80 °C for 3 h. After the reaction, the mixture
was cooled to room temperature in an ice water bath. The product
was washed with water and ethanol several times.
Synthesis of Pd Nanoplates on rGO Enclosed by {111}
Facets [{111}PdNP@rGO]. The synthesis of {111}PdNP@rGO was
similar to the synthesis of {100}PdSP@rGO, except for replacing KBr
with 74.55 mg of KCl.
Synthesis of Pd Nanocubes (Pd Cube). Pd nanocubes were
prepared according to previous studies.
29,30
Typically, PVP (105 mg),
AA (60 mg), and KBr (600 mg) were dissolved in 8.0 mL of water
and preheated in air under stirring at 80 °C for 10 min. Then, an
aqueous Na2PdCl4solution (3 mL, 57 mg of Na2PdCl4) was added.
After the vial had been capped, the reaction was allowed to proceed at
80 °C for 3 h.
Synthesis of rGO. The synthesis of rGO followed the typical
procedure but with the absence of Na2PdCl4.
Material Characterization. Fluorescence spectra were carried
out on a Shimadzu RF-6000 spectrofluorometer (Japan). UV−vis
absorption spectra were measured with a UV-8000 spectrophotom-
eter (Metash, China). The size and morphology of the products were
characterized by using a JEM-1400 transmission electron microscope
operated at 100 kV. High-resolution transmission electron microscopy
(HRTEM) images and the corresponding selected area electron
diffraction (SAED) patterns were both recorded on a JEM-2010
(HR) microscope operated at 200 kV. The thickness of the Pd
nanoplates was characterized by atomic force microscopy (AFM)
(Nanoscopt 8 Multimode Scanning Probe microscope (Vecco))
operating in contact mode with standard silicon nitride tips at room
temperature under ambient conditions.
Oxidase-like Activity of {100}PdSP@rGO. Twenty microliters
of TMB (10 mM) was mixed with 20 μL of {100}PdSP@rGO (0.313
mg/mL) in acetate buffer (20 mM, pH 4.4, 960 μL). After incubation
at 45 °C for 30 min, absorption spectra of the resultant solutions were
recorded.
Colorimetric Detection of ACP Activity. Typically, 20 μLof
{100}PdSP@rGO (0.313 mg/mL), 100 μL of acetate buffer (200
mM, pH 4.8), and 746 μL of ultrapure water were thoroughly mixed
in 1.5 mL centrifuge tubes. Then, 14 μL of AAP (10 mM), 100 μLof
ACP with various activities, and 20 μL of TMB (10 mM) were
introduced. The absorption spectra of resultant solutions were
measured after the reaction at 37 °C for 35 min.
■RESULTS AND DISCUSSION
Synthesis and Characterization of Pd Nanomaterials.
2D ultrathin Pd nanoplates enclosed by either {100} or {111}
facets were prepared by reduction of Na2PdCl4with AA at 80
°C in an aqueous solution containing annealed GO and KBr
(or KCl), as described previously.
28
Due to the abundant
carbonyl groups on the surface of annealed GO and Br−ion
selective adsorption on {100} facets, Pd nanoplates with
exclusively {100} facets could be produced [{100}PdSP@
rGO]. When replacing KBr with KCl, irregular Pd nanoplates
enclosed with {111} facets on rGO were generated
[{111}PdNP@rGO], while, in the absence of GO, we can
obtain the Pd nanocubes instead of Pd nanoplates. rGO could
be synthesized in the absence of a Pd salt precursor. The
morphologies of the Pd nanomaterials were initially
determined by TEM. As demonstrated in Figure 1A,
{100}PdSP@rGO presents a uniform square-like morphology
with an average edge length of ca. 200 nm and 3 nm in
thickness (Figures S1 and S2). The HRTEM image (inset at
the bottom left of Figure 1A) indicates that nanoplates are
single crystals with well-defined fringes. A lattice spacing of
0.20 nm can be attributed to the {100} facets of Pd with an fcc
(face-centered cubic) structure, similar to the observation in a
previous study.
25
Figure 1B shows the TEM image of Pd
nanoplates enclosed with {111} facets. Each nanoplates display
clear lattice fringes with interplanar spacings of 0.22 and 0.24
nm, corresponding to the {111} basal planes and 1/3 {422}
planes of the {111}PdNP@rGO, respectively. Figure 1C,D
ACS Applied Materials & Interfaces Research Article
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47565
shows that well-defined Pd nanocubes and rGO nanosheets
were successfully obtained.
Oxidase-like Activities of Pd Nanomaterials. Enzyme-
like catalytic abilities of Pd nanomaterials were first assessed
based on the example of {100}PdSP@rGO by using TMB,
ABTS, and OPD as typical chromogenic substrates. As shown
in Figure 2A, in the presence of {100}PdSP@rGO, solutions of
TMB, ABTS, and OPD correspondingly change from colorless
to blue, bluish green, and yellow, with the corresponding
maximum absorbance centered at around 652, 416, and 448
nm, respectively. In contrast, the colors of these chromogenic
substrates change negligibly in the absence of {100}PdSP@
rGO. The results indicates that {100}PdSP@rGO performs
oxidase-mimicking activity. Moreover, it was found that rGO
itself cannot trigger the oxidation of TMB (Figure 2B). Thus,
the oxidase-like activity of {100}PdSP@rGO exclusively arises
from the Pd nanoplates. The catalytic activity of {100}PdSP@
rGO is concentration-, pH-, and temperature-dependent
(Figure S6). The absorbance at 652 nm gradually increases
with increasing the concentration of {100}PdSP@rGO (Figure
S6A). The optimal pH value and temperature are found to be
4.4 and 45 °C, respectively (Figure S6B,C).
The catalytic mechanism was clarified through a series of
experiments. The {100}PdSP@rGO-catalyzed oxidization of
TMB apparently slows down under N2-saturated conditions
(Figure S7), which reveals the indispensable role played by
dissolved O2. Previous studies have shown that Pd-catalyzed
reactions like alkyne hydrogenation, formic acid electro-
oxidation, and O2activation are facet-sensitive.
30−34
As is
known to us, in many O2-involved chromogenic reactions, O2
is activated to generate highly reactive oxygen species (ROS),
where the adsorption of O2is a key step. Due to the different
atomic arrangements and charge states of catalyst surfaces,
surface facets can make an enormous impact on the O2
adsorption process.
34,35
To elucidate this, catalytic activities
of {100}PdSP@rGO and {111}PdNP@rGO were investigated
and compared by taking TMB as a chromogenic reagent. It is
obvious that the absorbance of the {100}PdSP@rGO-TMB
system is significantly higher (about 10.27 times) than that of
the {111}PdNP@rGO-TMB system after incubation at 45 °C
for 30 min (Figure 2B and Figure S8), indicating that
{100}PdSP@rGO possesses much higher oxidase-like activity
than {111}PdNP@rGO under the same experimental con-
Figure 1. (A−D) Representative TEM images of (A) {100}PdSP@
rGO, (B) {111}PdNP@rGO, (C) Pd nanocubes, and (D) rGO. The
insets in (A)−(C) are the HRTEM images of the corresponding Pd
nanomaterials.
Figure 2. (A) UV−vis absorption spectra of the 6.25 μg/mL {100}PdSP@rGO-catalyzed oxidation of 0.2 mM (a) TMB, (b) ABTS, and (c) OPD.
(B) UV−vis absorption spectra of (a) TMB, (b) rGO-TMB, (c) {100}PdSP@rGO-TMB, (d) {111}PdNP@rGO-TMB, and (e) Pd cube-TMB
solutions. Incubation at 45 °C for 30 min. The Pd content of each nanomaterial was fixed at the same level. (C) Michaelis−Menten kinetics for the
oxidation of TMB catalyzed by {100}PdSP@rGO, {111}PdNP@rGO-TMB, and Pd cube at room temperature. (D) Absorbance of the
{100}PdSP@rGO-TMB system in the presence of different scavengers. For isopropanol, tryptophan, and 4-benzoquinone: (a−e) 0, 0.2, 0.4, 0.6,
and 0.8 mM. For CAT: (a−e) 0, 1, 2, 3, and 4 U/mL. (E) UV−vis absorption spectra of NBT, {100}PdSP@rGO, and NBT+{100}PdSP@rGO.
(F) Oxidase-like activities of various Pd nanomaterials in catalyzing the oxidation of TMB.
ACS Applied Materials & Interfaces Research Article
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ditions. Figure 2C displays the divergence between the
behaviors of {100}PdSP@rGO and {111}PdNP@rGO.
Typical Michaelis−Menten curves can be obtained for both
{100}PdSP@rGO and {111}PdNP@rGO in a certain TMB
concentration range. To quantify the catalytic efficiency and
the affinity toward TMB, steady-state kinetic parameters,
including Michaelis−Menten constant (Km) and maximum
initial velocity (Vmax), are calculated by using double reciprocal
plots (Figure S9). Kmmeasured for {100}PdSP@rGO (0.093
mM) is lower than that of {111} PdNP@rGO (0.117 mM),
revealing that {100}PdSP@rGO exhibits a higher affinity for
TMB. Moreover, it was found that the Vmax value of TMB
oxidation catalyzed by {100}PdSP@rGO is more than three
times higher than that catalyzed by {111}PdNP@rGO,
demonstrating that {100}PdSP@rGO has excellent catalytic
efficiency. Thus, it can be concluded that {100}-faceted
PdSP@rGO could serve as an ideal candidate toward an O2-
involved chromogenic reaction. The superior catalytic property
can be attributed to the more highly active {100} facets.
Xiong’s group has suggested that surface facets can tune the
adsorption state and thus activation of O2on the Pd surface.
34
Molecular O2is better activated on Pd{100} because of the
large O−O bond length and the significant electron transfer
from the Pd surface to adsorbed O2. This could be the
principal reason for the much higher catalytic efficiency of
{100}PdSP@rGO than {111}PdSP@rGO.
Besides the surface facet effect, the substantially enhanced
oxidase-mimicking activity of {100}PdSP@rGO may also
originate from their 2D structure effect. The 2D Pd
nanostructure has a larger specific surface area and more
exposed high surface-active Pd atoms,
28
which will greatly
facilitate O2activation and activity enhancement. To confirm
this, the oxidase-like activity of Pd nanocubes is also analyzed.
As demonstrated in Figure 2B and Figure S8, the absorbance
of the {100}PdSP@rGO-TMB system is about 1.67 times
higher than that of the Pd cube-TMB system, and the Vmax
value displayed by {100}PdSP@rGO is approximately 1.54
times higher than that of Pd nanocubes (Figure 2C). These
results imply that the superior catalytic activity of
{100}PdSP@rGO originates not only from the highly active
{100} facets but also from the unique 2D structure.
To better understand the catalytic mechanism of Pd
nanoparticles, the type of generated ROS evolved from O2
was analyzed and determined. In principle, several possible
ROS could be involved in this catalytic reaction, including
superoxide anion (O2·−), hydroxyl radicals (·OH), singlet
oxygen (1O2), and hydrogen peroxide (H2O2). The system was
first examined using the scavenger mechanism. Isopropanol, 4-
benzoquinone, tryptophan, and CAT were used as the
scavengers for ·OH, O2·−,1O2, and H2O2, respectively.
36−38
As shown in Figure 2D and Figure S10A−C, isopropanol,
tryptophan, and CAT have little effects on the {100}PdSP@
rGO-catalyzed TMB oxidation, indicating that ·OH, 1O2, and
H2O2cannot be produced in this reaction. On the contrary, 0.6
mM 4-benzoquinone can almost completely inhibit the
catalytic reaction (Figure 2D and Figure S10D), implying
that O2·−is the main ROS formed on the surface of
{100}PdSP@rGO and is responsible for the TMB oxidation.
To further confirm the production of O2·−, NBT, which can
specifically react with O2·−to form blue-colored formazan,
39
was adopted as a colorimetric probe. As shown in Figure 2E, an
absorption peak centered at 680 nm emerged after introducing
{100}PdSP@rGO to NBT, suggesting the generation of O2·−.
Reasonably, the catalytic mechanism is proposed as follows
(Figure 2F): Dissolved O2, as an electron acceptor, is adsorbed
onto the surface of Pd nanomaterials and subsequently forms
O2·−on the Pd surfaces; then, the strong oxidizing O2·−
intermediate can oxidize TMB to form a blue product. The
order of catalytic efficiency is as follows: {100}PdSP@rGO >
Pd cube > {111}PdNP@rGO. For the sake of good detection
performance, {100}PdSP@rGO is employed for the subse-
quent assay of ACP activity due to its higher oxidase-like
activity.
ACP Detection Based on AA-Inhibited Oxidation of
TMB. AA is a powerful antioxidant due to its robust
reducibility. The O2·−adsorbed on the Pd surface with strong
oxidation ability can be consumed by AA efficiently, which will
directly obstruct the oxidation of TMB. Moreover, AA can
efficiently reduce the oxidized TMB (oxTMB) extremely
rapidly (Figure S11). As a result, by reacting with adsorbed
O2·−and reducing the oxTMB, AA can severely suppress the
{100}PdSP@rGO-catalyzed oxidation of TMB (Scheme 1).
The results in Figure 3 show that the absorbance of resultant
oxTMB solution progressively decreases with increasing the
concentration of AA from 0 to 40 μM.
In fact, besides being used as a target analyte, AA can
transmit signals in several analytical systems of bio-enzymes.
Inspired by the fact that ACP catalyzes the hydrolysis of AAP
into AA, a colorimetric ACP activity assay by using AAP as the
substrate is designed herein (Scheme 1). As well recognized,
the pH optimum and temperature optimum for ACP are 4−7
and 37 °C, respectively. Additionally, the {100}PdSP@rGO-
catalyzed chromogenic reaction of TMB works well in the pH
range of 3.0−5.8 and at 37 °C(Figure S6B,C). So, we intend
to conduct the ACP-catalyzed hydrolysis of AAP, the
{100}PdSP@rGO-triggered oxidation of TMB, and the
inhibition of {100}PdSP@rGO’s oxidase-like activity by
generating AA in one step. This makes the ACP analysis
procedure more simple and quick. The results in Figure 4A
verify the feasibility of our assumption. Incubating ACP-AAP
with PdSP@rGO-TMB at 37 °C for a certain time can
obviously inhibit the {100}PdSP@rGO-catalyzed oxidation of
TMB. Single ACP or AAP has no significant effect on this
chromogenic reaction.
To improve the performance of this ACP assay, detection
conditions like incubation time, pH, and AAP concentration
have been optimized. The performance of the system on ACP
detection is assessed by ΔA(ΔA=A0−A, where A0and A
correspond to the absorbance at 652 nm in the absence and
presence of ACP, respectively). The ΔAvalue increases
gradually in the first 35 min and then levels off(Figure S12A),
indicating that the reaction can be completed in 35 min. Thus,
Scheme 1. ACP Activity Detection Based on AA Controlled
Catalytic Activity of {100}PdSP@rGO
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35 min is the optimum incubation time for this ACP assay. As
acidic condition is propitious to the {100}PdSP@rGO-
catalyzed chromogenic reaction and the ACP-catalyzed
hydrolysis of AAP, pH within the range of 3.0−6.5 is
investigated. Maximal ΔAis achieved at pH 4.8 (Figure
S12B), which is adopted in the following experiments. For an
enzyme assay, both dynamic range and sensitivity are substrate
concentration-dependent. A low AAP concentration may result
in a narrow linear range. A high APP concentration does not
facilitate improvement of sensitivity and may exceed instru-
ment detection range. Considering these factors, 140 μM APP
was selected for further study (Figure S12C).
To investigate the specificity, the sensing system is
challenged with other nonspecific proteins/enzymes, including
BSA, trypsin, GOx, pepsin, lysozyme, pancreatin, and ALP.
The colorimetric results unambiguously illustrate that, except
ACP, none of these proteins/enzymes can induce distinct
colorimetric signals and interfere in the recognition response of
ACP under the optimized conditions (Figure 3B).
With ascertained experimental conditions and good
selectivity, the sensitivity of this colorimetric ACP assay is
analyzed. As demonstrated in Figure 4C, with increasing the
ACP activity from 0 to 10 mU/mL, UV−vis spectra decrease
progressively due to the increased concentrations of in situ
formed AA. In the ACP activity range of 0.01−6.0 mU/mL, a
splendid linear relationship between ΔAand ACP activities is
obtained with R2=0.992(Figure 4D). The calculated
detection limit is 8.3 μU/mL (3σ/S). The sensitivity is
superior to most previously reported ACP assays (Table S1).
The practicability and reliability of our proposed ACP
activity assay are assessed by detecting ACP activity in diluted
human serum samples (1%). Two serum specimens obtained
from the Hospital of University of Jinan are spiked with 2.0
and 4.0 mU/mL ACP. As shown in Table 1, satisfactory
recoveries between 94.1 ±1.45% and 103.1 ±3.15% are
obtained, indicating high accuracy of detecting ACP in
complex real samples.
Figure 3. (A) UV−vis absorption spectra of the analytical solution with various concentrations of AA. (B) Plots of absorbance at 652 nm versus AA
concentration. {100}PdSP@rGO, 6.25 μg/mL; TMB, 0.2 mM; pH 4.8 (20 mM); 37 °C, 35 min.
Figure 4. (A) Colorimetric response of the {100}PdSP@rGO-TMB solution containing (a) none, (b) AAP, (c) ACP, and (d) AAP-ACP. (B)
Absorbance of the analytical system toward ACP (8 mU/mL) and other unspecific proteins/enzymes (20 μg/mL). (C) UV−vis absorption spectra
with various activities of ACP. (D) Plots of ΔAversus ACP activities. AAP, 140 μM; {100}PdSP@rGO, 6.25 μg/mL; TMB, 0.2 mM; pH 4.8 (20
mM); 37 °C, 35 min.
Table 1. ACP Determination in Spiked Serums (1%)
samples added (mU/mL) found (mU/mL, n= 3) recovery (%, n=3)
A 2.0 1.882 ±0.029 94.1 ±1.45
4.0 3.965 ±0.054 99.1 ±1.35
B 2.0 2.012 ±0.015 100.6 ±0.75
4.0 4.125 ±0.126 103.1 ±3.15
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■CONCLUSIONS
In summary, we demonstrate that the oxidase-like activities of
Pd-based nanomaterials can be finely controlled by simulta-
neously tuning their facets and structures. Due to the 2D
structure with more active sites exposed on the surface and the
facet effect, {100}PdSP@rGO exhibits superior catalytic
performance for the oxidization reaction of TMB. Inspired
by the ACP-triggered hydrolysis of the substrate AAP and the
product AA controlled catalytic activity of {100}PdSP@rGO, a
colorimetric assay for ACP activity determination with high
selectivity and sensitivity is thus reasonably designed. This
assay is straightforward, sensitive, and selective enough for a
serologic test profiting from the easily detectable signal output
and simple procedures, the amplification of colorimetric signals
by nanozymes, and the intrinsic specificity of the enzymatic
reaction. We believe that this study will contribute to the better
rational design of other nanozymes with improved perform-
ance. It also paves a new avenue for developing nanozyme-
participant optical assays in a facile one-step manner.
■ASSOCIATED CONTENT
*
SSupporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsami.9b16279.
TEMandAFMimagesof{100}PdSP@rGO,
{111}PdSP@rGO, and Pd nanocubes; effects of catalyst
concentration, temperature, and pH on the oxidization
of TMB; effect of N2saturation on the catalytic activity
of {100}PdSP@rGO; comparison of oxidase-like
activities of various Pd nanomaterials; double reciprocal
plots; effect of different scavengers on the catalytic
activity of {100}PdSP@rGO; AA-medicated reducing of
oxTMB; optimization of reaction time, pH, and APP
concentrations; and comparison of various ACP assays
(PDF)
■AUTHOR INFORMATION
Corresponding Authors
*E-mail: mse_jiangyy@ujn.edu.cn (Y.J.).
*E-mail: mse_luyz@ujn.edu.cn (Y.L.).
*E-mail: weichen@ciac.ac.cn (W.C.).
ORCID
Yizhong Lu: 0000-0002-0914-2780
Wei Chen: 0000-0001-5700-0114
Author Contributions
∥
C.C. and W.L. contributed equally to this work.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
This work was supported by the National Natural Science
Foundation of China (21904048, 21902062, 21902061, and
21705056), the Young Taishan Scholars Program
(tsqn201812080), the Natural Science Foundation of
Shandong Province (ZR2019YQ10, ZR2018BB057,
ZR2017MB022, and ZR2018PB009), and the Open Funds
of the State Key Laboratory of Electroanalytical Chemistry
(SKLEAC201901).
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