Proton-Regulated Catalytic Activity of Nanozymes for Dual-Modal Bioassay of Urease Activity

Article abstract of DOI:10.1021/acs.analchem.1c01999

Benefiting from the merits of high stability and superior activity, nanozymes are recognized as promising alternatives to natural enzymes. Despite the great leaps in the field of therapy and colorimetric sensing, the development of highly sensitive nanozyme-involved photoelectrochemical (PEC) biosensors is still in its infancy. Specifically, the investigation of multifunctional nanozymes facilitating different catalytic reactions remains largely unexplored due to the difficulty in synergistically amplifying the PEC signals. In this work, mesoporous trimetallic AuPtPd nanospheres were synthesized with both efficient oxidase and peroxidase-like activities, which can synergistically catalyze the oxidation of 4-chloro-1-naphthol to produce benzo-4-chlorohexadienone precipitation on the surface of photoactive materials, and thus lead to the decreased photocurrent as well as increased charge-transfer resistance. Inspired by the proton-dependent catalytic activity of nanozymes, a self-regulated dual-modal PEC and electrochemical bioassay of urease activity was innovatively established by in situ regulating the activity of AuPtPd nanozymes through urease-mediated proton-consuming enzymatic reactions, which can remarkably improve the accuracy of the assay. Meanwhile, the determination of urease activity in spiked human saliva samples was successfully realized, indicating the reliability of the biosensor and its application prospects in clinical diagnosis.

Full text of DOI:10.1021/acs.analchem.1c01999

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Article
Proton-Regulated Catalytic Activity of Nanozymes for Dual-Modal
Bioassay of Urease Activity
Mingwang Liu, Guojuan Chen, Ying Qin, Jinli Li, Liuyong Hu,* Wenling Gu,* and Chengzhou Zhu*
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ABSTRACT: Benefiting from the merits of high stability and
superior activity, nanozymes are recognized as promising alternatives
to natural enzymes. Despite the great leaps in the field of therapy and
colorimetric sensing, the development of highly sensitive nanozyme-
involved photoelectrochemical (PEC) biosensors is still in its
infancy. Specifically, the investigation of multifunctional nanozymes
facilitating different catalytic reactions remains largely unexplored
due to the difficulty in synergistically amplifying the PEC signals. In
this work, mesoporous trimetallic AuPtPd nanospheres were
synthesized with both efficient oxidase and peroxidase-like activities,
which can synergistically catalyze the oxidation of 4-chloro-1-
naphthol to produce benzo-4-chlorohexadienone precipitation on
the surface of photoactive materials, and thus lead to the decreased
photocurrent as well as increased charge-transfer resistance. Inspired
by the proton-dependent catalytic activity of nanozymes, a self-regulated dual-modal PEC and electrochemical bioassay of urease
activity was innovatively established by in situ regulating the activity of AuPtPd nanozymes through urease-mediated proton-
consuming enzymatic reactions, which can remarkably improve the accuracy of the assay. Meanwhile, the determination of urease
activity in spiked human saliva samples was successfully realized, indicating the reliability of the biosensor and its application
prospects in clinical diagnosis.
enzymatic biosensors, which combine the selectivity of
enzymes and the sensitivity of PEC bioanalysis, are deemed
to promote their further advancements.38,39 However, natural
enzymes usually suffer from the disadvantages of high cost,
poor stability, and storage difficulties, which limit their
widespread applications.40−44 Moreover, enzyme immobiliza-
tion on the surface of a photoelectrode will inevitably hinder
the electron transfer and mass exchange during the PEC
process due to the insulating characteristics of natural
enzymes. To overcome these shortcomings, nanozymes, as
promising artificial enzymes, have been developed as
alternatives to natural enzymes due to low cost and high
stability.15,45−50 For example, ZnFe2O4 nanozymes,47 silver
iodide chitosan nanoparticles,45 and Au@CuS-GR48 were used
as peroxidase mimics to construct PEC biosensors. Despite
the great advantages of PEC nanozymatic biosensors, the
investigation of multifunctional nanozymes, which can
INTRODUCTION
■
Urease, as the earliest discovered nickel metalloenzyme, has
attracted extensive research interest for a long time since
being separated from the crystalline protein.1,2 Urease plays a
critical role in various metabolisms of organisms, which can
catalyze the decomposition of urea into ammonia and CO2.3
Many urinary and gastrointestinal diseases including cancer
and pyelonephritis are closely related to urease activity,4 and
the sensing of urease activity has been used to diagnose some
diseases.5 Noteworthily, the urease activity in the oral cavity is
recognized as an important diagnostic biomarker for early
disease diagnosis.6 Up to date, various methods such as
fluorescence,7 ELISA,8 electrochemistry,9 and colorimetric10,11
were applied for the analysis of urease. Nevertheless, the
development of an advanced functional dual-modal photo-
electrochemical (PEC) and electrochemical (EC) sensing
platform remains meaningful for urease detection. Recently,
PEC analysis, as a novel and promising analysis technique,
exhibits the merits of low cost, simple operation, antiback-
ground interference, high sensitivity, and miniaturization, and
provides great opportunities for fast and real-time detec-
tion.12−34 To realize a highly sensitive PEC biosensing, some
effective signal amplification strategies have been developed,
such as enzymatic reactions,35 the steric effect,13 energy
transfer,36 and the quenching effect.37 Among them, PEC
Received: May 11, 2021
Accepted: June 29, 2021
Published: July 9, 2021
https://doi.org/10.1021/acs.analchem.1c01999
© 2021 American Chemical Society
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facilitate different varieties of catalytic reactions, remains
largely unexplored due to the difficulty in synergistically
amplifying the PEC signals. Furthermore, dual-modal sensing
platforms with advanced functions are greatly expected to
improve the accuracy of an assay. Therefore, finely modulating
the functionality of nanozymes will provide great prospects for
further exploring their corresponding applications in PEC and
EC dual-modal biosensing.
In this work, trimetallic AuPtPd mesoporous nanospheres
were rationally designed, which displayed both excellent
oxidase and peroxidase-like activities. After the immobilization
of AuPtPd nanozymes on the surface of photoactive BiVO4,
no obvious recession of photocurrent was observed,
manifesting the superiority of nanozymes in PEC biosensing.
Owing to the remarkable oxidase and peroxidase-like activities
of AuPtPd nanozymes, biocatalytic precipitation could be
synergistically produced on the surface of a photoelectrode to
trigger the significant photocurrent variation and increased
charge-transfer resistance (Rct) in the presence of H2O2 and 4-
chloro-1-naphthol (4-CN). Inspired by the proton-dependent
catalytic activity of nanozymes, a self-regulated dual-modal
bioassay based on PEC and EC methods for the evaluation of
urease activity was innovatively designed by in situ regulating
the activity of AuPtPd nanozymes through urease-mediated
proton-consuming enzymatic reactions (Scheme 1). As
was collected by centrifugation at 8000 rpm for 5 min and the
residual Pluronic F127 was removed by five consecutive
washing/centrifugation cycles with ultrapure water. After
vacuum drying at 60 °C for 12 h, AuPtPd nanospheres
were obtained.
Oxidase and Peroxidase-like Activities of the AuPtPd
Nanospheres. To verify the oxidase-like activity of AuPtPd
nanospheres, 3,3′,5,5′-tetramethylbenzidine (TMB) was used
as a chromogenic substrate. Specifically, 10 μL of 0.5 mg
mL−1 of AuPtPd was added to 150 μL of acetate buffer
solution with different pH values (pH range from 3 to 9), to
which 50 μL H2O and 50 μL TMB (1 mM) were added. After
being kept at 37 °C for 15 min, the resulting mixed solution
was investigated by UV−vis scanning using a multimode
reader. Similarly, 50 μL of 10 mM H2O2 was used instead of
water to evaluate the peroxidase-like activity of AuPtPd. The
oxidase and peroxidase-like activities were assessed by
monitoring the absorbance value at 652 nm.54 The steady-
state kinetic assays of oxidase-like were performed at 37 °C in
NaAc buffer containing 0.1 mg mL−1 AuPtPd and various
concentrations of TMB. All measurements were monitored by
the absorbance change at 652 nm. The kinetic parameters
were calculated using the Michaelis−Menten equation: ν =
Vmax × [S]/(Km + [S]), where ν is the initial velocity, [S]
represents the substrate concentration, Vmax is the maximal
velocity, and Km represents the Michaelis−Menten constant.
Synthesis of BiVO4 and BiVO4−AuPtPd Photoelectr-
odes. BiVO4 photoelectrodes were prepared according to the
previous report.55 Preparation of the electroplating solution:
the pH of 50 mL mixed solution containing 0.04 M Bi(NO3)3·
5H2O and 0.4 M KI was adjusted to 1.7 with HNO3; then 20
ml of the ethanol solution containing 0.23 M benzoquinone
was slowly added to the solution. After the pH became stable,
a typical three-electrode system of an ITO working electrode
(WE), an Ag/AgCl (4 M KCl) reference electrode (RE), and
a platinum counter electrode (CE) was used for electro-
deposition with a CHI 760D EC workstation at −0.1 V versus
Ag/AgCl under vigorous magnetic stirring. The duration of
the electrodeposition was around 5 min and the current
density−time curve of BiOI during electrodeposition is
displayed in Figure S1. Afterward, 50 mL dimethyl sulfoxide
solution containing 0.08 M VO (acac)2 was evenly dropped
onto the surface of the BiOI electrode (1.0 × 1.0 cm2) before
thermal treatment in air at 450 °C for 2 h (ramp rate 2 °C
min −1). The BiVO4 photoelectrodes can be obtained after
being placed in 1 M NaOH solution to remove excess V2O5.
The electrodes were then thoroughly rinsed with ultrapure
water, blow-dried with nitrogen, and stored in a dry place for
further use. 30 μL AuPtPd aqueous solution (0.2 mg mL−1)
was dropped onto the surface of the BiVO4 electrode, and
then vacuum dried at 50 °C for 6 h to obtain BiVO4−AuPtPd
photoelectrodes.
Scheme 1. Mechanism of Dual-Modal PEC and EC
Biosensor Based on AuPtPd Nanozymes and BiVO4 for
Urease Detection
expected, the multifunctional AuPtPd nanozyme-based dual-
modal biosensor exhibits outstanding selectivity, good
repeatability, and promising applicability to real sample
detection.
EXPERIMENTAL SECTION
■
Synthesis of Mesoporous Trimetallic AuPtPd Nano-
spheres. The mesoporous trimetallic AuPtPd nanospheres
were synthesized by a typical method with some modifica-
tion.51−53 First, 4.8 mL of 0.02 M K2PtCl4, 1.6 mL of 0.02 M
Na2PdCl4 (Na2PdCl4 was prepared by dissolving PdCl2 in
sodium chloride solution), 1.6 mL of 0.02 M HAuCl4, and
100.0 μL of 6.0 M HCl were mixed to form a homogeneous
solution. Then, 80.0 mg of Pluronic F127 was added to the
above solution under sonication. After the Pluronic F127 was
completely dissolved in the solution, 8.0 mL of the 1.5 mM
ascorbic acid (AA) solution was added to the solution
immediately. The mixed solution was kept in a water bath at
40 °C for 6 h under continuous stirring. Finally, the product
Dual-Modal PEC and EC Detection of Urease
Activity. First, 240 μL of the mixed solution including 50
μL urea solution (20 mM), 90 μL urease reaction buffer
(acetate buffer, 5 mM, pH 4.0), and 100 μL various amounts
of urease (0−3000 mU mL−1) were added in a 96-well plate
and incubated at 37 °C for 30 min. Then, 50 μL 4-CN (1
mM) and 20 μL H2O2 (10 mM) were added to the above
solution and mixed evenly. Finally, the above mixture (30 μL)
was dropped on the surface of the BiVO4−AuPtPd photo-
electrode and incubated at 37 °C for 30 min. PEC
measurements were carried out using a CHI 842D EC
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Figure 1. SEM images of BiVO4 (A) and BiVO4−AuPtPd (B). (C) XRD patterns of BiVO4, AuPtPd, and BiVO4−AuPtPd. (D) XPS survey
spectrum of BiVO4−AuPtPd. (E) Bi 4f in BiVO4−AuPtPd. (F) V 2p in BiVO4−AuPtPd.
workstation in a standard three-electrode configuration with
the as-synthesized electrode used as the WE, a platinum wire
as the CE, and an Ag/AgCl (saturated with KCl) electrode as
the RE. All PEC measurements were performed at phosphate
buffer solution (PBS, 0.1 M, pH 7.0) containing 1.5 mM AA
at 0 V. Rct was obtained by electrochemical impedance
spectroscopy (EIS) under the same reaction conditions. EIS
was measured on a CHI 660E EC workstation (Shanghai
Chenhua Apparatus Corporation, China) with a three-
electrode system in 1 M KCl solution containing the 5 mM
K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture as the electrolyte
in a frequency range of 0.1 Hz−100 kHz.
Analysis of Urease in the Real Human Saliva Sample.
Saliva samples were collected from a healthy volunteer with
informed consent. Samples were 10-fold diluted with ultrapure
water to avoid interference, and the pH of the saliva sample
was adjusted to 7.0. Then, the saliva sample was used to
prepare urease solution in different concentrations with a
volume of 100 μL. Finally, the spiked samples can be obtained
by adding 90 μL reaction buffer (acetate buffer solution, 5
mM, pH 4.0) into the above solution.
photoelectrodes show a nanoporous and worm-like morphol-
ogy, which favors charge transport. In addition, AuPtPd
nanozymes exhibit a relatively uniform size and shape,
showing a porous spherical structure with a diameter of
about 150 nm (Figure S3). As for BiVO4−AuPtPd (Figure
1B), the morphology of AuPtPd was well maintained and
evenly dispersed on BiVO4. Furthermore, the elemental
composition and spatial distribution were studied by energy-
dispersive X-ray (EDX) element mapping analysis (Figure
S4). The existence of Bi, V, O, Au, Pt, and Pd was confirmed
in the BiVO4−AuPtPd composite material, which demon-
strated the successful attachment of AuPtPd onto the surface
of BiVO4.
XRD was used to analyze the phase structure of these
samples (Figure 1C), which indicated the successful synthesis
of BiVO4 (JCPDS14-0688) and trimetallic AuPtPd (Au:
JCPDS-04-0784 and JCPDS-04-0784; Pt: JCPDS-04-0802;
Pd: JCPDS 46-1043). Noteworthily, the lattice mismatch
ratios of Pt/Pd and Pt/Au are 0.77 and 4.08%, respectively. It
is difficult to distinguish the peaks of Pt and Pd in the XRD
pattern, while the peaks of Au and Pt−Pd can be easily
recognized.52,56 To further confirm the surface chemical
compositions and electronic states, the BiVO4−AuPtPd
electrodes were investigated by XPS (Figure 1D). Consistent
with the results of EDX element mapping analysis (Figure
S5), the general survey XPS further indicated that BiVO4−
AuPtPd contained the elements of Bi, V, O, Au, Pt, and Pd. In
addition, Figure 1E,F shows the high-resolution XPS spectra
of Bi 4f and V 2p, respectively. The peaks located at 159.2,
RESULTS AND DISCUSSION
■
Design and Characterization of the Resultant
Materials. Noble metal-based nanomaterials have been
demonstrated promising enzyme-like catalytic activity. To
realize a nanozyme with multifunctionality, trimetallic AuPtPd
mesoporous nanospheres were rationally designed by virtue of
the synergistic effect of different noble metals. Moreover,
BiVO4 was chosen as the photoactive material to guarantee a
remarkable photocurrent resulting from its good light-
harvesting ability. The morphologies and structures of
AuPtPd, BiVO4, and BiVO4−AuPtPd were investigated
utilizing scanning electron microscopy (SEM), X-ray
diffraction (XRD), and X-ray photoelectron spectroscopy
(XPS). The BiOI electrode was synthesized by electro-
deposition, and then annealed in air to prepare the BiVO4
electrode. According to the digital photographs shown in
Figure S2, the successful formation of BiVO4 photoelectrodes
with a typical color was clearly observed. As can be seen from
the SEM image (Figure 1A), the as-prepared BiVO4
164.5, 516.6, and 524.2 eV can be assigned to Bi 4f7/2, Bi 4f5/2
,
V 2p3/2, and V 2p1/2, respectively, and are the characteristic
peaks of Bi and V in BiVO4.57
Then, the PEC activities of the as-prepared photoelectrodes
were measured by the instantaneous photocurrent response in
0.1 M PBS (pH 7.0) at a constant potential of 0 V under
visible light (λ ≥ 420 nm) irradiation. To alleviate charge
recombination, AA was added as an effective hole scavenger to
quickly consume holes in BiVO4. As shown in Figure 2A, the
bare ITO electrode exhibited a negligible photocurrent, while
the BiVO4 and BiVO4−AuPtPd modified electrodes show a
relatively strong anodic photocurrent. To optimize the
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indicating the n-type characteristic of BiVO4. In addition, the
flat band potential (Efb) can be obtained as 0.28 eV according
to the intercept on the abscissa (Figure S7).
Evaluation of Oxidase and Peroxidase-like Activities
of AuPtPd Nanozymes. The oxidase and peroxidase-like
activities of AuPtPd nanozymes were investigated with a
typical chromogenic reaction, during which the oxidation of
TMB into oxTMB was triggered with or without H2O2. As
shown in Figure 2E, in the presence of AuPtPd and TMB, a
strong absorption peak was observed at 652 nm. After further
addition of H2O2, the absorption peak intensity at 652 nm
increased, while no distinct color change was observed for the
individual TMB or TMB + H2O2 system, demonstrating the
superb oxidase and peroxidase-like features of AuPtPd
mesoporous spheres. Owing to the oxidase and peroxidase-
like activities of AuPtPd nanozymes, the oxidation of 4-CN
would be catalyzed to form insoluble benzo-4-chlorohexadie-
none (4-CD) precipitates on the photoelectrode surface,
leading to a large interfacial resistance and a suppressed
photocurrent. As exhibited in Figure 2F, a significant variation
in the photocurrent could be realized upon the addition of
H2O2, demonstrating the advantages of multifunctional
nanozymes in synergistic signal amplification. Inspired by
the proton-dependent regulatory mechanism for natural
enzymes, the pH-regulated catalytic activities of AuPtPd
nanozymes were investigated. As depicted in Figure S8A,B,
the superb oxidase and peroxidase-like properties of AuPtPd
nanozymes were achieved at a pH value of 4, and their
activities decayed rapidly when the concentration of protons
decreased. Therefore, the oxidase and peroxidase-like activities
of AuPtPd nanozymes can be rationally tailored by proton-
consuming enzyme-catalyzed bioreactions, which in turn
influence the formation of bioprecipitation via the oxidation
of 4-CN and subsequently alter the photoresponse and Rct of
the photoelectrode. To better understand the oxidase-like
activity of AuPtPd nanozymes, the steady-state kinetics was
investigated. Typical Michaelis−Menten curves for AuPtPd
nanozymes were obtained over a certain range of TMB
concentrations (Figure S9). The Km and Vmax values of
AuPtPd nanozymes were determined to be 6.28 mM and 3.41
× 10−7 M s−1 for TMB, respectively.
Performance of the Dual-Modal Biosensor for
Urease Detection. Urease, as a typical proton-consuming
enzyme, can catalyze the decomposition of urea to produce
ammonia, making it feasible to modulate the catalytic activities
of AuPtPd nanozymes and achieve the dual-modal detection
of urease activity. First, the reaction time and temperature
were optimized to guarantee the best catalytic activity of
urease (Figure S10A,B) and AuPtPd nanozymes (Figure
S11A,B), and a reaction time of 30 min and a reaction
temperature of 37 °C were chosen as the optimal conditions.
As shown in Figure 3A, the photocurrent of the pristine
BiVO4−AuPtPd photoelectrode is 22.5 μA. In the absence of
urease, no proton was consumed, and AuPtPd nanozymes
exhibited the best activity to catalyze the oxidation of 4-CN
into 4-CD precipitation to significantly inhibit the photo-
current (Figure 3A). However, the catalytic activity of AuPtPd
nanozymes will be depressed and a higher photocurrent was
observed along with the proton-consuming bioreaction in the
presence of urease (Curease = 160 mU mL−1) (Figure 3A).
Therefore, it can be concluded that the pH-regulated PEC
performance of the BiVO4−AuPtPd photoelectrode was
suitable for the sensitive detection of urease. Figure 3B
Figure 2. (A) Photocurrent of bare ITO, BiVO4, and BiVO4−
AuPtPd in 0.1 M PBS (pH 7) at 0 V under visible light. (B) EIS
Nyquist plots of BiVO4 and BiVO4−AuPtPd in 1 M KCl solution
containing 5 mM [Fe(CN)6]3−/4−. (C) UV−vis diffuse reflectance
spectra of BiVO4, AuPtPd, and BiVO4−AuPtPd. (D) Tauc plots of
BiVO4 and BiVO4−AuPtPd. (E) UV−vis absorption spectra of TMB,
TMB + AuPtPd, TMB + H2O2, and TMB + AuPtPd + H2O2. (F)
The influence of H2O2 on photocurrent in the PEC analysis system.
photocurrent of BiVO4−AuPtPd, the amounts of AuPtPd
nanozymes were studied and a concentration of 0.2 mg mL−1
was selected as the optimal concentration for the following
experiment (Figure S6A). As a result, AuPtPd nanozymes
exhibit a slight effect on the photocurrent of BiVO4. In
addition, the superior stability of photocurrent based on
BiVO4−AuPtPd photoactive materials under 400 s on/off
light irradiation has also been verified (Figure S6B).
EIS measurement was employed to further investigate the
influence of AuPtPd nanozymes on the charge-transfer
resistance of the photoelectrode.58 Generally speaking, the
Nyquist graph is composed of a semicircle and a linear part.13
Among them, the semicircle diameter represents the Rct value
between the interface of the electrode and the electrolyte. As
shown in Figure 2B, the Rct value of BiVO4−AuPtPd was
changeless compared with that of BiVO4, indicating that the
introduction of AuPtPd nanozymes has no effect on the
charge transfer.
The light absorption properties of BiVO4, AuPtPd, and
BiVO4−AuPtPd were measured with the UV−vis diffuse
reflectance (Figure 2C). As expected, AuPtPd nanozymes with
metallic properties exhibit no obvious absorption edge, while
the absorption edges of both the BiVO4 and BiVO4−AuPtPd
samples are about 520 nm. The band gap energies (Eg) of
both BiVO4 and BiVO4−AuPtPd were almost unchanged,
which were 2.34 eV (Figure 2D). According to the Mott−
Schottky curve of BiVO4, the tangent slope is positive,
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photocurrent change induced by urease, which indicated the
good selectivity of the BiVO4−AuPtPd-based PEC sensing
platform for the urease detection. In addition, the stability
(Figure 3E) for 400 s of switching light irradiation and the
reproducibility (Figure 3F) of the prepared PEC biosensor
were studied at a urease concentration of 60 mU mL−1.
Notably, the photocurrents were almost constant during the
measurements, which indicates the satisfactory stability and
reproducibility of the sensor.
We also further verified the feasibility of detecting urease
through the EIS method. The experimental procedure was
similar to the PEC sensing system for urease. As shown in
Figure 4A, the Nyquist plot of the pristine BiVO4−AuPtPd
Figure 3. (A) Photocurrent response of the pristine BiVO4−AuPtPd
and BiVO4−AuPtPd photoelectrodes incubated at various concen-
trations of urease (0 and 160 mU mL−1). (B) Photocurrent of the
PEC sensing system with different concentrations of urease (0, 10,
15, 30, 60, 160, 320, 750, 1500, and 3000 mU mL−1). (C) A linear
plot of photocurrent response versus the logarithm of the urease
concentration (10−750 mU mL−1). (D) Selectivity of the PEC
biosensor for urease detection (Curease = 60 mU mL−1, I0 is the initial
photocurrent responses of BiVO4−AuPtPd). (E) Time-based photo-
current responses of the PEC sensing system (Curease = 60 mU mL−1)
with light on and off. (F) Repeatability of a PEC sensor (Curease = 60
mU mL−1).
Figure 4. (A) EIS Nyquist plots of the BiVO4−AuPtPd photo-
electrode with various concentrations of urease (0, 60, and 750 mU
mL−1) in 1 M KCl solution containing 5 mM [Fe(CN)6]3−/4−. (B)
EIS Nyquist plots of the BiVO4−AuPtPd photoelectrode with various
concentrations of urease (0, 10, 30, 60, 160, 320, 750, 1500, and
3000 mU mL−1). (C) A linear plot of Rct versus the logarithm of the
urease concentration (10−750 mU mL−1). (D) Selectivity of the
biosensor for urease (160 mU mL−1) detection (Rct is the initial
charge-transfer resistance of BiVO4−AuPtPd).
presents the photocurrents of the BiVO4−AuPtPd-based PEC
sensing system toward different concentrations of urease (10−
3000 mU mL−1). As expected, the photocurrent of BiVO4−
AuPtPd photoelectrodes gradually increases along with the
continuously increasing urease concentration, while it remains
almost changeless at higher concentrations than 750 mU
mL−1 (Figure 3B). As a result, the linear relationship between
the PEC response and the logarithm of the urease
concentration was realized in the range of 10−750 mU
mL−1 (Figure 3C), which can be described using the formula
of I (μA) = 8.65 log Curease − 3.53 (R2 = 0.998). The limit of
detection (LOD, S/N = 3) was calculated to be 8.4 mU mL−1.
To evaluate the selectivity of the as-prepared PEC biosensor
for urease, the photocurrent responses toward some other
potentially common compounds that may be present in
samples were investigated. Common biomolecules such as uric
acid, lactose, and glucose (50 μM), amino acids such as
leucine, L-serine, and phenylalanine (50 μM), and biological
enzymes such as laccase, glucose oxidase, tyrosinase, and
alkaline phosphatase (2000 mU mL−1) were selected as
interferers. As displayed in Figure 3D, the effect of the added
interferences is ignorable compared with the significant
photoelectrode displays a small semicircle with an Rct value of
about 120 Ω (black curve). In the absence of urease, the
largest Rct was observed, which was in accordance with its
smallest photocurrent. As expected, the Rct value decreased
significantly with the increase of concentrations of urease.
Notably, the Rct value of BiVO4−AuPtPd photoelectrodes
gradually decreased along with the continuously increasing
urease concentration, which remains almost constant at higher
concentrations than 750 mU mL−1 (Figure 4B).
As a result, the linear relationship between the Rct value and
the logarithm of the urease concentration was realized in the
range of 10−750 mU mL−1 (Figure 4C), which can be
described by the formula Rct (μA) = −96.9 log Curease + 400.1
(R2 = 0.995). The LOD was calculated to be 6.6 mU mL−1.
Similarly, the Rct inhibition from several interferences is
ignorable in contrast with urease, which declares the excellent
selectivity of the proposed biosensor based on BiVO4−
AuPtPd. Notably, Table S1 shows the comparison of our
results with those of the previously reported method for the
determination of urease, indicating that the results obtained
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using the biosensor based on the BiVO4−AuPtPd photo-
electrode are among the best-known results.
Furthermore, the applicability of this urease sensing system
was assessed in the human saliva samples by the standard
addition method. The recovery tests were performed, and the
relative standard deviations (RSDs) were acquired from three
samples. As shown in Table 1, the recoveries varied from 96.0
detection by PEC; comparison of the results obtained
from the two methods of PEC and EC; and comparison
of different methods for urease detection (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
Liuyong Hu − Hubei Engineering Technology Research Center
of Optoelectronic and New Energy Materials, Hubei Key
Laboratory of Plasma Chemistry and Advanced Materials,
School of Materials Science and Engineering, Wuhan
Institute of Technology, Wuhan 430205, P. R. China;
Email: huly@wit.edu.cn
Wenling Gu − Key Laboratory of Pesticide and Chemical
Biology of Ministry of Education, International Joint
Research Center for Intelligent Biosensing Technology and
Health, College of Chemistry, Central China Normal
University, Wuhan 430079, P. R. China; Email: wlgu@
mail.ccnu.edu.cn
Chengzhou Zhu − Key Laboratory of Pesticide and Chemical
Biology of Ministry of Education, International Joint
Research Center for Intelligent Biosensing Technology and
Health, College of Chemistry, Central China Normal
University, Wuhan 430079, P. R. China; orcid.org/
0000-0003-0679-7965; Email: czzhu@mail.ccnu.edu.cn
Table 1. Determination of Urease in Human Saliva Samples
spiked
found
recovered
(%)
RSD
(%)
sample
(mU mL−1
)
(mU mL−1
)
I
II
III
10.0
100.0
500.0
9.6 0.07
97.5 0.47
507.2 1.65
96.0
97.5
101.4
3.73
3.16
1.42
to 101.4% and the RSDs ranged from 1.42 to 3.73%,
indicating the reliability of the biosensor and its potential
application in clinical diagnosis. Moreover, urease with
concentrations of 20, 50, 100, 200, and 500 mU mL−1 were
selected to study the PEC and EC performance of the system.
The test procedure was the same as the previous procedure
for measuring urease activity. The resulting photocurrent value
was substituted into the linear equation to obtain the urease
concentration. A good linear correlation (R2 = 0.992) was
observed between the results obtained from the PEC and the
EC method (Figure S12), indicating the acceptable reliability
and accuracy of the dual-modal biosensor.
Authors
Mingwang Liu − Key Laboratory of Pesticide and Chemical
Biology of Ministry of Education, International Joint
Research Center for Intelligent Biosensing Technology and
Health, College of Chemistry, Central China Normal
University, Wuhan 430079, P. R. China
Guojuan Chen − Key Laboratory of Pesticide and Chemical
Biology of Ministry of Education, International Joint
Research Center for Intelligent Biosensing Technology and
Health, College of Chemistry, Central China Normal
University, Wuhan 430079, P. R. China
Ying Qin − Key Laboratory of Pesticide and Chemical Biology
of Ministry of Education, International Joint Research Center
for Intelligent Biosensing Technology and Health, College of
Chemistry, Central China Normal University, Wuhan
430079, P. R. China
Jinli Li − Key Laboratory of Pesticide and Chemical Biology of
Ministry of Education, International Joint Research Center
for Intelligent Biosensing Technology and Health, College of
Chemistry, Central China Normal University, Wuhan
430079, P. R. China
CONCLUSIONS
■
In this work, multifunctional AuPtPd nanozymes were
reasonably designed and synthesized with outstanding oxidase
and peroxide-like activities. Based on the urease-mediated
proton-consuming enzymatic reactions, a dual-modal PEC and
EC biosensor for the evaluation of urease activity was
innovatively constructed by in situ regulating the synergistic
catalytic activity of AuPtPd nanozymes. Based on these
strategies, the as-prepared biosensor shows excellent selectiv-
ity, good repeatability, and high stability. Meanwhile, the
determination of urease activity in spiked human saliva
samples was successfully realized, indicating the reliability of
the biosensor and its application prospects in clinical
diagnosis. This work sheds light on the superiority of
multifunctional nanozymes in synergistic amplification and
opens up a new avenue for building a sensitive biosensing
platform with nanozymes.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.analchem.1c01999.
■
sı
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.analchem.1c01999
Notes
Materials and reagents; instrument; current−time curve
for electrodeposition of BiOI; digital images of the BiOI
and BiVO4 photoelectrodes; SEM image of AuPtPd;
elemental mapping images of BiVO4−AuPtPd; EDX
element mapping analysis; effect of AuPtPd on the
photocurrent of BiVO4−AuPtPd; PEC stability of the
BiVO4−AuPtPd electrode; Mott−Schottky curve of
BiVO4; oxidase and peroxidase-like activities of the
AuPtPd nanozymes; steady-state kinetic assay of TMB
oxidation by AuPtPd nanozymes; optimum reaction
time and temperature for urease incubation in a 96-well
plate; optimal reaction time and temperature for urease
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge the financial support by
the National Natural Science Foundation of China (nos.
22074049 and 22004042), the Natural Science Foundation of
Hubei Province (2020CFB276), the Fundamental Research
Funds for the Central Universities (nos. CCNU20QN007 and
CCNU20TS013), and the Program of Introducing Talents of
Discipline to Universities of China (111 program, B17019).
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Anal. Chem. 2021, 93, 9897−9903

Analytical Chemistry
pubs.acs.org/ac
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