In Situ Exsolution of Noble-Metal Nanoparticles on Perovskites as Enhanced Peroxidase Mimics for Bioanalysis

Enhanced Peroxidase Mimics for Bioanalysis

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In Situ Exsolution of Noble-Metal Nanoparticles on Perovskites as

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Xiaoqian Jiang, Xiaoyu Wang, Anqi Lin, and Hui Wei*

Cite This: Anal. Chem. 2021, 93, 5954 −5962

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sı Supporting Information

ABSTRACT: Various transition-metal oxide (TMO)-based nanoma-

terials have been explored as peroxidase mimics. However, the moderate

peroxidase-like activity of TMOs limited their widespread use.

Decorating highly active noble-metal nanozymes on the surface of

TMOs can not only enhance the peroxidase-like activity of TMOs but

also prevent the small-sized metal nanoparticles (NPs) from aggregation.

Herein, in situ exsolution of noble-metal NPs (i.e., Ir and Ru) from A-

site-deﬁcient perovskite oxides (i.e., chemical formula La B B′ O₃₋_δ,

.9 0.9 0.1

B = Mn/Fe, B′ = Ir/Ru) under a reducing atmosphere was achieved for

preparing noble-metal NPs/perovskite composites. The exsolved NPs

were socketed on the surface of parent perovskite oxides, which

signiﬁcantly enhanced the stability of metal NPs. In addition, the

peroxidase-like activity of perovskite oxides increased remarkably after

NPs egress. We then used the optimized Ir/LMIO with high stability and excellent peroxidase-like activity to develop a colorimetric

assay for the determination of alkaline phosphatase (ALP). Beneﬁting from the remarkable peroxidase-like activity of Ir/LMIO, the

sensing platform exhibited a wide linear range. The practical application of the colorimetric sensing method was demonstrated by

detecting the ALP in serum samples. This work not only provides new insights into the synthesis of highly active peroxidase-like

nanozymes but expands their applications for constructing a high-performance biosensing platform.

INTRODUCTION

peroxidase-like activity of perovskite oxides was still inferior to

noble-metal-based nanozymes.

Previous studies demonstrated that metal NPs (e.g., Ru and

■

Nanozymes are nanomaterials with enzyme-like catalytic

activities.

Various types of enzymes such as oxidase,

Ir) with small sizes exhibited excellent peroxidase-like

peroxidase, catalase, superoxide dismutase, and hydrolase

have been mimicked using diﬀerent kinds of nanomaterials.

Among these nanozymes, nanomaterials with peroxidase-like

activity have received great attention due to their great promise

in biosensing, bioimaging, and nanomedicine.

workers ﬁrst discovered that Fe O nanoparticles (NPs) could

mimic a peroxidase that were then used as alternatives to

horseradish peroxidase for immunoassays. Inspired by this

research, various transition-metal oxides (TMOs) were ex-

plored as peroxidase mimics.

3,24

−7

activity.

These noble-metal-based nanozymes were usually

24,25

synthesized by a wet chemical reduction process.

control the size and morphology of metal nanozymes, capping

or encapsulating agents were employed during the synthesis.

However, the capping agents on metal NP surface may block

access to the catalytic sites, resulting in the decreased

peroxidase-like activity. In addition, despite the presence of

capping agents, metal-based nanozymes with small sizes could

easily aggregate during practical catalysis process.

−13

Yan and co-

5−18

However, most of the metal

oxides nanozymes have moderate catalytic activity. Therefore,

eﬃcient strategies are needed to design highly active TMO-

based peroxidase mimics. We have recently demonstrated that

Recently, the exsolution based on in situ growth of metal

NPs from a parent perovskite has been demonstrated as an

eﬀective method for preparing metal NPs/perovskite oxide

ABO -type perovskite oxides (where A is a rare-earth or

26−29

composites.

The target transition metal (B′) was ﬁrst

alkaline-earth metal and B is a transition metal) are good

candidates for this purpose due to their unique physical and

chemical properties, ﬂexible structures, and adjustable

incorporated into the lattice of A-site deﬁciency perovskite

9−22

Received: February 17, 2021

Accepted: March 23, 2021

Published: April 2, 2021

components.

We showed that the e occupancy is a

predictive descriptor for the rational design of perovskite

oxide-based nanozymes. LaNiO₃₋_δ, optimized based on the e_g

occupancy, possessed a peroxidase-like activity 1−2 orders of

magnitude higher than that of other representative carbon-

based and metal oxide-based nanozymes. Even so, the

2021 American Chemical Society

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Anal. Chem. 2021, 93, 5954−5962

Analytical Chemistry

Article

oxide to form A₁₋_xB₁₋_yB′ O₃₋_δ. Subsequently, the target

stirring and then evaporated to form gel. The gel was further

heated at 180 °C for 5 h to form the solid precursors. The

obtained solid precursor was decomposed at 400 °C for 2 h to

remove the organic components and to obtain foam

precursors. The foam precursors were further annealed in air

at 900 °C for 5 h with a ramp rate of 5 °C/min to obtain the

pristine perovskite (denoted as LMIO, LMRO, LFIO, and

transition metal (B′) was exsolved to form ﬁnely dispersed

30−33

metal NPs under a reducing atomsphere.

Therefore, we

envision that in situ exsolution of metal NPs on perovskites will

not only improve the peroxidase-like activity of perovskite

oxides but also prevent metal NPs from aggregation during the

catalytic process.

Here, we proposed a strategy involving in situ exsolution of

Ir or Ru NPs on the surface of perovskite oxides by A-site

defects as driving forces under a reducing atmosphere.

LFRO) and control samples (LaMnO₃and LaFeO ),

respectively. Then, the pristine perovskite powders were

reduced at 650 °C for 5 h in 5% H /Ar to obtain the ﬁnal

La Mn Ir O

(LMIO), La Mn Ru O

(LFIO), and La Fe Ru O

(LMRO),

(LFRO)

samples, namely, Ir/LMIO, Ru/LMRO, Ir/LFIO, and Ru/

LFRO.

0.9 0.1 3−δ

0.9

0.1 3−δ

La Fe Ir O

.9 0.9 0.1 3−δ

0.9 0.9 0.1 3−δ

were used as parent materials to in situ exsolve Ir or Ru NPs on

perovskite oxides surface to form Ir/LMIO, Ru/LMRO, Ir/

Instrumentation. Transmission electron microscopy

(TEM) images were recorded on a JEOL JEM-2100

transmission electron microscope (JEOL, Japan) at an

acceleration voltage of 200 kV. High-resolution transmission

electron microscopy (HRTEM), high-angle annular dark-ﬁeld

scanning TEM (HAADF-STEM), and the corresponding

energy-dispersive spectroscopy (EDS) elemental mappings

were performed on an FEI Titan G2 60-300 TEM equipped

with an acceleration voltage of 300 kV and an EDS detector,

respectively. Powder X-ray diﬀraction (XRD) patterns were

obtained on a Rigaku Ultima X-ray diﬀractometer using Cu Kα

radiation. X-ray photoelectron spectra (XPS) were collected

using a PHI 5000 VersaProbe (Ulvac-Phi, Japan). UV−visible

absorption spectra were collected using a spectrophotometer

(TU-1900, Beijing Purkinje General Instrument Co. Ltd.,

China). The absorption in a 96-well plate at 652 nm was

recorded by a SpectraMax M2e microplate reader (Molecular

Devices).

LFIO, and Ru/LFRO, respectively. LaMnO was ﬁrst chosen

as the parent material due to its relatively good peroxidase-like

activity and stability. Furthermore, to prove the universality of

this method, LaFeO was also used as a parent material for in

situ exsolution of Ru and Ir NPs. The peroxidase-mimicking

activity of Ir/LMIO, Ru/LMRO, Ir/LFIO, and Ru/LFRO

exhibited about 10, 7, 50, and 30 times higher than their

corresponding parent perovskite oxides.

Finally, Ir/LMIO with the highest peroxidase-like activity

was employed to develop a reliable bioanalysis platform for the

determination of alkaline phosphatase (ALP). ALP is present

in the body ﬂuids and tissues of human, which is a crucial

diagnostic indicator of many diseases. Its abnormal level is

mainly related to liver disease, biliary obstruction, metabolic

34−36

disease, scurvy, bone diseases, etc.

The hydrolysis of

phosphoryl esters could be catalyzed by ALP to produce

37,38

ascorbic acid (AA),

which exhibited a competitive eﬀect

Peroxidase-like Activity Measurements. The perox-

idase-like activity was measured using TMB as a substrate.

Steady-state kinetics assays were conducted at 25 °C in a 96-

well plate and recorded in a microplate reader. For the kinetics

assays of nanozymes, acetic acid−sodium acetate buﬀer (pH

4.5, 0.2 M) was used as the reaction buﬀer and 10 μg/mL of

nanozymes were used. Kinetics data were obtained by

changing the concentration of one substrate (i.e., H O or

on the reaction between H O and TMB. Based on this, we

have built a colorimetric sensing platform to evaluate the ALP

activity. Beneﬁting from the remarkable peroxidase-like activity

of Ir/LMIO, the colorimetric method exhibited a wide linear

range and a low detection limit.

EXPERIMENTAL SECTION

■

TMB) and keeping that of the other constant. The absorption

at 652 nm within 5 min was monitored continuously. The

Chemicals and Materials. Lanthanum nitrate (La(NO ) ·

H O), iron nitrate (Fe(NO ) ·9H O), iridium chloride

^kⁱⁿ^e^tⁱ^c^s^p^a^r^a^m^e^t^e^r^s⁽ⁱ^.^e^.^,^vmax and K ) were calculated by ﬁtting

hydrate (IrCl ·xH O), ruthenium chloride hydrate (RuCl ·

the reaction velocity values and the substrate concentrations to

the Michaelis−Menten equation as follows

xH O), 3,3′,5,5′-tetramethylbenzidine (TMB), trypsin, gly-

cine, glutamic acid, sarcosine, and glucose oxidase (GOx) were

purchased from Aladdin Chemical Reagent Co., Ltd.

Manganese nitrate (Mn(NO ) ·4H O) was obtained from

v [S]

max

v =

Beijing Inokai Technology Co., Ltd. Hydrogen peroxide

H O ), ethylene glycol, ascorbic acid (AA), and citric acid

K + [S]

(1)

(

were purchased from Sinopharm Chemical Reagent Co., Ltd.

Glucose was purchased from Nanjing Chemical Reagent Co.,

Ltd. Fructose was purchased from Shandong Xiya Reagent Co.,

Ltd. Ascorbic acid 2-phosphate (AAP), alkaline phosphatase

where v is the initial reaction velocity, v_m_a_xis the maximal

reaction velocity, K is the Michaelis constant, and [S] is the

substrate concentration.

Eﬀect of AA on the Peroxidase-like Activity of Ir/

LMIO. H O (20 mM), TMB (500 μM), Ir/LMIO (2 μg/mL),

(

ALP), lysozyme, and bovine serum albumin (BSA) were

purchased from Sigma-Aldrich. Human serum albumin (HSA)

and lactate oxidase (LOx) were purchased from Shanghai

Yuanye Bio-Technology Co., Ltd. All aqueous solutions used

in the experiments were prepared with deionized water (18.2

MΩ·cm, Millipore).

and AA with various concentrations (0−200 μM) in 0.2 M

acetic acid−sodium acetate buﬀer (pH 4.5) were mixed. Then,

100 μL of the reaction solution was added into a 96-well plate

and the absorption at 652 nm was recorded in a microplate

reader for 10 min at 25 °C.

Colorimetric Detection of ALP. Colorimetric ALP

activity assay was performed as follows. First, 0.2 M Tris-

HCl (pH 8.0), AAP (0.2 mM), and ALP (with diﬀerent

activities ranging from 0 to 200 U/L) were mixed and

incubated in a 37 °C water bath for 40 min. Then, 0.2 M acetic

acid−sodium acetate buﬀer (pH 4.5) was added to stop the

Catalyst Preparations. The pristine perovskites were

synthesized via a sol−gel method. The respective stoichio-

metric metal nitrates (3 mmol in total) and citric acid (12

mmol) were dissolved in 100 mL of H O, then 1.5 mL of

ethylene glycol was added under stirring. The resulting

transparent solutions were treated at 90 °C with suﬃcient

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Figure 1. (a) Schematic illustration of the synthesis process of in situ exsolution of noble-metal NPs on perovskite. (b) TEM image of LMIO. (c, d)

TEM images of Ir/LMIO. (e) HAADF-STEM image and EDS elemental mapping of Ir/LMIO. (f) HRTEM images of Ir/LMIO. (g) XRD patterns

of LMIO and Ir/LMIO. (h, i) XPS proﬁles of LMIO and Ir/LMIO in Ir 4f spectrum and Mn 2p spectrum, respectively.

catalytic reaction. Subsequently, 2 μg/mL of Ir/LMIO (or

Millipore) for 3 min at 14 000 rpm. Finally, 100 μL of the

LMIO and LaMnO ), 0.5 mM TMB, and 90 mM H O were

above solution, 0.5 mM TMB, and 90 mM H O were added

added into the reaction solution. Then, 100 μL of the reaction

solution was added into the 96-well plate and the absorption at

into 0.2 M acetic acid−sodium acetate buﬀer (pH 4.5) (ﬁnal

total volume was 1 mL). Then, 100 μL of the reaction solution

was added into a 96-well plate and the absorption at 652 nm

was recorded in a microplate reader for 10 min at 25 °C.

52 nm was recorded in a microplate reader for 10 min at 25

To investigate the selectivity of ALP detection, 100 U/L

GOx, lysozyme, trypsin, and LOx, 500 μM metal ions (K ,

RESULTS AND DISCUSSION

■

Na , and Ca ), 100 μM amino acids (glycine, glutamic acid,

and sarcosine), fructose, glucose, 3 μM HSA, and BSA as

interference were determined.

Synthesis and Characterization of the Perovskite-

Based Nanozymes. In situ exsolution of Ir or Ru NPs on the

surface of perovskite oxides was achieved by A-site defects as

driving forces under a reducing atmosphere (Figure 1a).

To detect ALP activity in serum samples, 25 μL of serum

5%) was spiked with diﬀerent concentrations of ALP solution

(

and diluted to 500 μL using 0.2 M Tris-HCl buﬀer (pH 8.0).

Then, 0.2 mM AAP was added into the spiked samples as

described above. After incubation at 37 °C for 40 min, the

mixture was ultraﬁltered to remove the interference with a

Microcon centrifugal ﬁlter device (Ultracel YM-30 membrane,

LaMnO perovskite was ﬁrst selected as the parent materials

because it not only possesses excellent peroxidase-like activity

but is stable upon annealing at a reducing atmosphere. To in

situ exsolve Ir nanoparticle, La Mn Ir O (LMIO)

0.9 0.1 3−δ

perovskite with A-site deﬁciency was synthesized, because A-

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Figure 2. (a) TEM image of LMRO. (b, c) TEM images of Ru/LMRO. (d) XRD patterns of LMRO and Ru/LMRO. (e, f) XPS proﬁles of LMRO

and Ru/LMRO in Ru 3p spectrum and Mn 2p spectrum, respectively.

δ+

site deﬁciency could serve as a driving force to trigger B-site

4f₇_/₂was ∼61.26 eV, which belonged to Ir , and no metallic Ir

was detected. After treatment at 650 °C under a forming gas of

5% H in argon, the Ir 4f was deconvoluted into two double

0,39

egress.

As shown in Figure 1b, LMIO prepared by a sol−

gel method exhibited a clean and smooth surface. However,

many ﬁnely dispersed NPs with diameters of 1−2 nm were

socketed on the surface of perovskite host after annealing

LMIO at a reducing atmosphere (Figure 1c,d). To decipher

the composition of the exsolved NPs, HAADF-STEM images

coupled with EDS element mapping were performed. As

presented in Figure 1e, after NPs exsolution, the uniform

distribution of La, Mn, and O in the parent perovskite was

observed. Most of surface Ir cations were reduced and became

NPs, while the concentration of ionic Ir near the surface of the

perovskite was negligible (Figure 1e). The HRTEM results in

Figure 1f exhibited a magniﬁed NP and perovskite matrix with

well-deﬁned lattice fringes. The NP with a d-spacing of 0.192

nm corresponded to cubic (space group Fm3m) Ir NPs

indexed at the (200) crystal planes, while the perovskite matrix

with a d-spacing of 0.276 nm corresponded to orthorhombic

7/2

δ+

peaks that can be assigned to Ir and Ir , with binding energies

of 59.68 and 60.49 eV, respectively, indicating that Ir NPs have

appeared on the surface of the perovskite matrix. The average

valence state of Mn decreased slightly after exsolution (Figure

1i). The above results demonstrated that in situ exsolution of

NPs from the parent perovskite could be an eﬀective approach

for synthesizing Ir NPs/perovskite composites.

To demonstrate the universality of in situ exsolution of

noble-metal NPs from parental perovskites, Ru was taken as an

another example due to its excellent peroxidase-like activity.

As shown in Figure 2a−c, metal NPs were embedded on the

surface of the perovskites when the La Mn Ru O

0.1 3−δ

0.9

(LMRO) was reduced at 5% H /Ar. In addition, HRTEM

images conﬁrmed that the NPs on the surface of perovskites

with a d-spacing of 0.205 nm corresponded to Ru NPs (space

group P63/mmc) indexed at the (101) crystal planes (Figure

2c). XRD results suggested that no other byproducts appeared

after exsolution of Ru NPs (Figure 2d). The unique binding

energy of the Ru 3p₃_/₂on LMRO was ∼463.83 eV,

demonstrating that only Ru was present in the pristine

perovskite matrix. After reduction, the Ru 3p₃_/₂of Ru NPs was

deconvoluted into two peaks, which belonged to Ru (461.6

eV) and Ru (463.17 eV), demonstrating that Ru NPs were

(

space group Pnma) LaMnO matrix indexed at the (002)

crystal planes. Moreover, powder X-ray diﬀraction (XRD) was

used to analyze the structure changes after NPs exsolution.

Before annealing at 5% H /Ar, LMIO presented typical

δ+

diﬀraction peaks of rhombohedral (JCPDS 82-1152) with a

space group of R3c. No obvious impurities were observed,

suggesting that Ir was successfully doped into the LaMnO₃

perovskite lattice. After reduction, the perovskite oxide was

δ+

subjected to a structural phase change (Figure 1g). The XRD

patterns of LMIO and Ir/LMIO suggested that the lattice’s

structure transformed from rhombohedral (JCPDS 82-1152)

to orthorhombic (JCPDS 72-0841) with a space group of

Pnma. No new peaks from byproducts appeared after reducing.

The peaks of Ir NPs were not observed in the XRD pattern,

which may be attributed to the fact that the size of Ir NPs was

too small to be detected by XRD. We further analyzed the

chemical valence states of the nanozymes by X-ray photo-

electron spectroscopy (XPS) measurements. As shown in

Figure 1h, for the pristine LMIO, the binding energy of the Ir

exsolved from the perovskite lattice (Figure 2e). In addition,

the average valence state of Mn decreased slightly after

exsolution (Figure 2f).

Other than LaMnO , we also demonstrated that the noble-

metal NPs could be also exsolved from other perovskite oxides.

LaFeO was selected as another host material for in situ

exsolution of Ru and Ir NPs. HRTEM, XRD, and XPS results

demonstrated that both Ir and Ru NPs can be successfully

exsolved on the surface of LaFeO perovskite matrix without

the destruction of the perovskite structures (Figures S1 and

S2 ).

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Figure 3. Peroxidase-like property of diﬀerent nanozymes. (a) UV−vis spectra of samples containing 1 mM TMB, 20 mM H O , and 10 μg/mL of

diﬀerent nanozymes in 0.2 M acetate buﬀer for 10 min. (b) Plots of the velocity of the reaction versus diﬀerent concentrations of H O with 1 mM

TMB and 10 μg/mL of diﬀerent nanozymes in 0.2 M acetate buﬀer for 5 min. Error bars indicate standard deviations of three independent

measurements. (c) Double-reciprocal plots of the velocity versus varying concentration of H O . Error bars indicate standard deviations of three

independent measurements. (d) Comparison of the peroxidase-like activities through v_m_a_x

Peroxidase-like Activity of Noble-Metal Nanopar-

ticle/Perovskite Composites. After successful synthesis of

Ir/LMIO, Ru/LMRO, Ir/LFIO, and Ru/LFRO, their perox-

idase-like activities were evaluated by monitoring the catalytic

oxidation of TMB in the presence of H O . As shown in Figure

Table 1. Comparisons of Kinetic Parameters between

Diﬀerent Peroxidase Mimics

substrate/H O₂

LaMnO₃LMIO Ir/LMIO LMRO Ru/LMRO

^vmax (10⁻M s⁻¹

)

21.84

11.52

44.29

14.15

LFIO

240.4

47.47

152.2

69.15

153.1

68.28

^Km (mM)

a, Ir/LMIO rapidly catalyzed the oxidation of TMB by H O ,

2 2

Ir/LFIO LFRO Ru/LFRO

producing an obviously blue-colored oxidation product with a

characteristic absorbance at 652 nm. However, the same

concentration of H O alone produced a negligible absorbance

^s^u^b^s^t^r^a^t^e^/^H^O2

^L^a^F^e^O3

max (10⁻M s⁻¹

)

2.699

32.45

3.468

14.42

139.0

52.33

7.819

27.46

82.24

93.58

(mM)

change. Moreover, in comparison to the parent LaMnO and

LMIO, Ir/LMIO had the highest catalytic eﬃciency of TMB

oxidation in the presence of H O , indicating that in situ

compared to LaMnO and LMIO, Ir/LMIO showed a much

exsolution of Ir NPs on the surface of LaMnO signiﬁcantly

higher v_m_a_xvalue when H O was used as the changing

enhanced the peroxidase-like activity of parental materials.

To investigate the kinetic process and quantify the

peroxidase-like activity of noble NPs/perovskite composites

after reduction, the steady-state kinetic measurements were

performed. The kinetic data were obtained by changing the

concentration of one substrate (e.g., H O or TMB) when

substrate (Figure 3d), indicating that the peroxidase-like

activity of LaMnO perovskite could be signiﬁcantly enhanced

by in situ exsolving Ir NPs. In addition, we also studied the

kinetic parameters of LMRO, LFIO, and LFRO before and

after exsolution. Typical Michaelis−Menten curves were

obtained in the suitable concentration range of H O (Figure

keeping the other’s concentration constant. The enzyme

kinetic parameters were obtained by Michaelis−Menten curves

and the Lineweaver−Burk plot (Tables 1 and S1). Typical

4a−c), which showed the same results as Ir/LMIO (i.e., after

the exsolution, the peroxidase-like activities were enhanced).

K and v values were obtained through the Lineweaver−

max

Michaelis−Menten curves of LaMnO , LMIO, and Ir/LMIO

Burk plot in Figure S3, which showed higher v values after

max

are shown in Figure 3b. The maximum initial velocity (v

)

exsolution (Figure 4d). We used the v_m_a_xvalue of the parent

perovskites as the standard to normalize; the performance

improvements of Ir/LMIO and Ru/LMRO are about 10 and 7

max

and Michaelis−Menten constant (K ) were obtained through

the double-reciprocal plots of initial reaction rates (Figure 3c).

The value of v_m_a_xshowed the maximum rate as a critical

indicator of the catalytic activity of the reaction. The value of

K_mis inversely proportional to the aﬃnity between the

nanozymes and the substrate. It was noteworthy that,

times higher than their parent material LaMnO , while the

performance improvements of Ir/LFIO and Ru/LFRO are

about 50 and 30 times higher than their parent material

LaFeO , respectively. Among them, Ir/LMIO has the best

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Figure 4. Steady-state kinetic assays of diﬀerent nanozymes. Plots of the velocity of the reaction versus diﬀerent concentrations of H O with 1 mM

TMB and 10 μg/mL diﬀerent nanozymes in 0.2 M acetate buﬀer for 5 min. Error bars indicate standard deviations of three independent

measurements. (a) Ru/LMRO, LMRO, and LaMnO . (b) Ir/LFIO, LFIO, and LaFeO . (c) Ru/LFRO, LFRO, and LaFeO . (d) Comparison of

the peroxidase-like activities through v_m_a_x

peroxidase-like activity due to the synergy between the highly

catalytic activity of host perovskite and the noble-metal NPs.

The kinetic data with TMB as a substrate are presented in

Figures S4 and S5 and Table S1, demonstrating that whether

H O or TMB as a substrate, the peroxidase-like activity of

perovskite oxides was signiﬁcantly improved after exsolution of

Ir or Ru NPs.

eﬀect on the reaction between H O and TMB (Figure 5a).

2 2

On the basis of this, we developed a colorimetric assay for ALP

by coupling the cascade reactions of AAP hydrolysis and the

nanozyme-catalyzed TMB oxidation (Figure 5a). As a proof of

concept, we ﬁrst investigated the inhibitory eﬀect of AA on the

TMB oxidation. As shown in Figures 5b and S7, the A − A

value decreased signiﬁcantly with increasing AA concentration

In addition, Ir NPs with an average size of 2 nm were

(where A and A are the absorbances of the system at 652 nm

synthesized by a wet chemical method. Both the catalytic

eﬃciency and stability of free Ir NPs and incorporated ones

were investigated. Our study demonstrated that the exsolution

had little eﬀect on the intrinsic catalytic activity of Ir NPs

after 5 min of the reaction and when the reaction just started,

respectively). The linearity range for AA was 1−100 μM with a

detection limit of 0.2 μM. Based on the above results, we

performed experiments to assess the feasibility for detecting

ALP activity. ALP or AAP exhibited a negligible eﬀect on the

TMB oxidation catalyzed by Ir/LMIO in the presence of H O

(

Tables 1 and S1 ). However, after storing for more than 6

months, the peroxidase-like activity of Ir/LMIO was only

reduced by 33%, while the activity of Ir NPs at the same mass

concentration changed signiﬁcantly with a 97% decrease

(Figure 5c). However, when ALP and AAP coexisted in the

reaction system, the absorbance of oxTMB at 652 nm was

substantially decreased because AAP was hydrolyzed to

generate AA in the presence of ALP. As shown in Figure 5d,

Figure S6 ). The above results demonstrated that in situ

exsolution of noble-metal NPs from perovskite oxide surface

can be a promising strategy that not only eﬀectively improves

the peroxidase-like activity of parental materials but also

enhances the stability of noble-metal NPs.

a good linear relationship between A − A and ALP

concentration was obtained. The linear range for ALP was

0.39−100 U/L, which was broader than most of other

methods (Table S2 ). In addition, the limit of detection

(LOD) of this sensing platform was as low as 0.39 U/L,

demonstrating its potential for ALP detection in real serum

samples. To demonstrate the unique advantages of Ir/LMIO,

Colorimetric Assay of ALP Activity. Alkaline phospha-

tase (ALP) is distributed at various levels in the body ﬂuids

and tissues of human, and its abnormal expression is closely

related to many diseases. Therefore, it is signiﬁcant to

construct a colorimetric strategy to detect ALP with high

sensitivity and selectivity in clinical diagnostics. ALP can

catalyze the hydrolysis of the orthophosphoric monoesters

LaMnO and LMIO were also used as peroxidase mimics for

ALP detection. For comparison, the linear ranges of the

LMIO/TMB/H O and LaMnO /TMB/H O sensing plat-

(

e.g., AAP) to produce ascorbic acid (AA). As a kind of

forms were 0.24−8 and 0.34−10 U/L (Figures 5e and S8 ),

respectively. The LODs were 0.24 and 0.34 U/L, respectively

radical chain-breaker antioxidants, AA exhibits competitive

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Figure 5. (a) Schematic illustration of a colorimetric platform for ALP activity assay. (b) Plot of A − A versus concentrations of AA of Ir/LMIO.

Inset: Linear plot of A − A versus concentrations of AA. (c) UV−vis absorption spectra of various reaction systems. (d) Linear plot of A − A

versus concentrations of ALP (0.39−100 U/L). (e) Plot of A − A versus concentrations of ALP of diﬀerent nanozymes. Error bars indicate

standard deviations of three independent measurements.

Table S3 ). The substantially increased detection linear range

Table 2. Results for the Determination of ALP in Serum

Samples

for Ir/LMIO sensing platform could be mainly attributed to

much higher peroxidase-like activity because other conditions

of three sensing platforms were kept identical. The enhanced

peroxidase-like activity of Ir/LMIO nanozyme makes the

colorimetric system have greater application potential.

added (U/L)

found (U/L)

recovery (%)

RSD (%, n = 3)

10.26

7.54

5.00

2.38

102.60

100.51

100

1.09

0.21

0.54

1.02

To evaluate the selectivity of the as-developed sensing

95.20

method for ALP detection, we investigated possible interfering

substances common in human serum. These include K , Na ,

.1%, suggesting that the proposed sensing system can

Ca , glucose oxidase (GOx), lysozyme, trypsin, lactate oxidase

LOx), HSA, BSA, fructose, glucose, glycine, glutamic acid,

accurately detect ALP in serum samples.

(

and sarcosine. Figure S9 shows that the interference of other

substances on the sensing platform was negligible, conﬁrming

that the developed sensing system has a satisfactory selectivity

toward ALP detection.

The ALP content in serum samples was measured to

demonstrate the practical application of the colorimetric assay.

As shown in Table 2, the recoveries for the four biological

samples were between 95.20 and 102.60% with RSD less than

CONCLUSIONS

■

In conclusion, a general and eﬀective in situ exsolution strategy

was developed to prepare highly active peroxidase-like

nanozymes. The in situ exsolution of Ir or Ru NPs from

noble-metal-substituted A-site-deﬁcient perovskites can not

only eﬀectively improve the peroxidase-like activity of parent

perovskite but also improve the stability of noble-metal NPs.

The obtained Ir/LMIO nanozyme has the highest peroxidase-

960

Anal. Chem. 2021, 93, 5954−5962

Analytical Chemistry

The Supporting Information is available free of charge at

Article

like activity compared with other exsolved nanozymes.

Through coupling Ir/LMIO nanozyme with ascorbic acid 2-

phosphate, a facile colorimetric assay for ALP activity detection

with high sensitivity and selectivity was developed. This study

not only provides a promising strategy to rationally design

nanomaterials with peroxidase-mimicking activity but also

develops a facile colorimetric assay to sensitively detect ALP

activity.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by the National Key R&D Program

of China (2019YFA0709200), National Natural Science

Foundation of China (21874067 and 21722503), the China

Postdoctoral Science Foundation (2019TQ0144 and

(

019M661786), CAS Interdisciplinary Innovation Team

JCTD-2020-08), PAPD Program, Open Funds of Key

Laboratory of Analytical Chemistry for Biology and Medicine

Wuhan University), Ministry of Education (ACBM2019001),

ASSOCIATED CONTENT

sı Supporting Information

https://pubs.acs.org/doi/10.1021/acs.analchem.1c00721.

■

(

the Innovation Foundation of Nanjing University, and

Fundamental Research Funds for the Central Universities

TEM images, PXRD patterns, XPS survey scans, kinetic

curves, absorption spectra, stability of free Ir NPs and Ir/

LMIO, comparisons of kinetic parameters between

diﬀerent peroxidase mimics, and comparisons of

analytical performance between diﬀerent peroxidase

mimics of this work in ALP detection (PDF )

(

021314380145).

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