Nanozymes Next Wave of Artificial Enzymes

SPRINGER BRIEFS IN MOLECULAR SCIENCE
Xiaoyu Wang
Wenjing Guo
Yihui Hu
Jiangjiexing Wu
Hui Wei
Nanozymes: Next
Wave of Artificial
Enzymes
12 3
SpringerBriefs in Molecular Science
More information about this series at http://www.springer.com/series/8898
Xiaoyu Wang Wenjing Guo
•
Yihui Hu Jiangjiexing Wu
•
Hui Wei
Nanozymes: Next Wave

of Artificial Enzymes
123
Xiaoyu Wang Jiangjiexing Wu
Department of Biomedical Engineering, College of Department of Biomedical Engineering, College of
Engineering and Applied Sciences, Collaborative Engineering and Applied Sciences, Collaborative
Innovation Center of Chemistry for Life Sciences, Innovation Center of Chemistry for Life Sciences,
Nanjing National Laboratory of Microstructures Nanjing National Laboratory of Microstructures
Nanjing University Nanjing University
Nanjing Nanjing
China China
Wenjing Guo Hui Wei

Department of Biomedical Engineering, College of Department of Biomedical Engineering, College of
Engineering and Applied Sciences, Collaborative Engineering and Applied Sciences, Collaborative
Innovation Center of Chemistry for Life Sciences, Innovation Center of Chemistry for Life Sciences,
Nanjing National Laboratory of Microstructures Nanjing National Laboratory of Microstructures
Nanjing University Nanjing University
Nanjing Nanjing
China China
Yihui Hu
Department of Biomedical Engineering, College of
Engineering and Applied Sciences, Collaborative
Innovation Center of Chemistry for Life Sciences,
Nanjing National Laboratory of Microstructures
Nanjing University
Nanjing
China
ISSN 2191-5407 ISSN 2191-5415 (electronic)

SpringerBriefs in Molecular Science
ISBN 978-3-662-53066-5 ISBN 978-3-662-53068-9 (eBook)
DOI 10.1007/978-3-662-53068-9
Library of Congress Control Number: 2016946947
© The Author(s) 2016

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Preface
This book is intended to describe the concepts, the up-to-date developments, and
the perspectives of the field of nanozymes that has been rapidly growing over the
past decades. Nanozymes are nanomaterials with enzymatic characteristics. As one
of the most exciting fields, the research of nanozymes lies at the interface of
chemistry, biology, materials, and nanotechnology.
It is counterintuitive to use nanomaterials to mimic natural enzymes since the
two seem to be very different from each other. A careful comparison, however,
would reveal that they share many features together. For examples, both of them
have nanoscaled sizes, irregular shapes, rich surface chemistry, etc. It is these
similarities that enable nanomaterials to imitate natural enzymes.
Due to the enormous amounts of literature published in the field, it is impossible
to provide a comprehensive description of nanozymes here. Instead, it aims to
provide a broad picture of nanozymes in the context of artificial enzyme research.
Representative examples are discussed to highlight the nanomaterials with enzyme
mimicking activities, their catalytic mechanisms, and their promising applications
in various areas, ranging from biosensing and cancer diagnostics to tissue engi-
neering and therapeutics.
Chapter 1 describes the brief history of nanozymes research in the course of
natural enzymes and artificial enzymes research. It also compares nanozymes with
natural enzymes and artificial enzymes to highlight their unique characteristics.
Chapters 2–5 discuss the different nanomaterials used for mimicking various natural
enzymes, from carbon-based (Chap. 2) and metal-based (Chap. 3) nanomaterials to
metal oxide-based nanomaterials (Chap. 4) and other nanomaterials (Chap. 5). In
each of these chapters, the nanomaterials’ enzyme mimetic activities, the catalytic
mechanisms, and the key applications are covered. In Chap. 6, the current chal-
lenges and future directions of nanozymes research are summarized, which if
achieved will help to fulfill the great potentials of nanozymes.
The purpose of this book is not only to provide insightful knowledge of nano-
zymes but also to attract more researchers into the field and to inspire them to
further broaden the field. Due to the importance of nanozymes and professional
v
vi Preface
writing with plenty of color illustrations and tables, this book should be an ideal
choice for readers from different areas, such as chemistry, materials, nanoscience
and nanotechnology, biomedical and clinical studies, environment, green chem-
istry, novel catalysts, etc.
I wish to express my appreciation to all the excellent scholars around the world
who have contributed and will continue to contribute to the fields of nanozymes.
I would also like to thank my lab members and my collaborators for their contri-
butions to this exciting field. I thank my advisors Profs. Erkang Wang, Xinghua
Xia, Yi Lu, and Shuming Nie for their guidance, support, and encouragement. I am
much indebted to June Tang for her patience during the writing of this book.
I thank Nanjing University, National Natural Science Foundation of China, 973
Program, Natural Science Foundation of Jiangsu Province, Shuangchuang Program
of Jiangsu Province, PAPD program, Fundamental Research Funds for Central
Universities, Six Talents Summit Program of Jiangsu Province, Open Funds of the
State Key Laboratory of Electroanalytical Chemistry, Open Funds of the State Key
Laboratory of Analytical Chemistry for Life Science, Open Funds of the State Key
Laboratory for Chemo/Biosensing and Chemometrics, and Thousand Talents
Program for Young Researchers for providing the academic environment and the
financial support of our research.
Nanjing Hui Wei

April 2016
Contents
1 Introduction to Nanozymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2 Carbon-Based Nanomaterials for Nanozymes . . . . . . . . . . . . . . . . . . . 7
2.1 Fullerene and Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.1.1 Fullerene and Derivatives as Nuclease Mimics . . . . . . . . . . 8
2.1.2 Fullerene and Derivatives as SOD Mimics . . . . . . . . . . . . . 9
2.1.3 Fullerene Derivatives as Peroxidase Mimics . . . . . . . . . . . . 12
2.2 Graphene and Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.2.1 Graphene and Its Derivatives as Peroxidase Mimics . . . . . . 12
2.2.2 Decorated Graphene (or Its Derivatives)
as Peroxidase Mimics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.3 Carbon Nanotubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.3.1 Carbon Nanotubes as Peroxidase Mimics . . . . . . . . . . . . . . 20
2.3.2 Carbon Nanotubes as Other Enzyme Mimics . . . . . . . . . . . 22
2.4 Other Carbon-Based Nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . 24
2.4.1 Other Carbon Nanomaterials as Peroxidase Mimics . . . . . . 24
2.4.2 Other Carbon Nanomaterials as SOD Mimics . . . . . . . . . . . 24
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3 Metal-Based Nanomaterials for Nanozymes . . . . . . . . . . . . . . . . .... 31
3.1 Metal Nanomaterials with Catalytic Monolayers (Type I). . . . .... 31
3.1.1 AuNPs Protected by Alkanethiol with Catalytic
Terminal Moieties . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 32
3.1.2 AuNPs Protected by Alkanethiol with Non-covalently
Assembled Catalytic Moieties . . . . . . . . . . . . . . . . . . . .... 37
3.1.3 AuNPs Protected by Thiolated Biomolecules . . . . . . . .... 39
vii
viii Contents
3.2 Metal Nanomaterials with Intrinsic Enzyme Mimicking

Activities (Type II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.2.1 Metal Nanomaterials as GOx Mimics . . . . . . . . . . . . . . . . . 40
3.2.2 Metal Nanomaterials as Multiple Enzyme Mimics . . . . . . . 41
3.2.3 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4 Metal Oxide-Based Nanomaterials for Nanozymes . . . . . . . . . . . . . . . 57
4.1 Cerium Oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.1.1 Cerium Oxide as SOD Mimics . . . . . . . . . . . . . . . . . . . . . . 58
4.1.2 Cerium Oxide as Catalase Mimics . . . . . . . . . . . . . . . . . . . 64
4.1.3 Cerium Oxide as Peroxidase Mimics . . . . . . . . . . . . . . . . . 66
4.1.4 Cerium Oxide as Oxidase Mimics . . . . . . . . . . . . . . . . . . . . 66
4.1.5 Cerium Oxide as Other Mimics . . . . . . . . . . . . . . . . . . . . . 67
4.2 Iron Oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
4.2.1 Iron Oxide as Peroxidase Mimics . . . . . . . . . . . . . . . . . . . . 68
4.2.2 Iron Oxide as Other Enzyme Mimics . . . . . . . . . . . . . . . . . 76
4.3 Other Metal Oxides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
4.3.1 Vanadium Oxide as Enzyme Mimics . . . . . . . . . . . . . . . . . 78
4.3.2 Cobalt Oxide as Enzyme Mimics . . . . . . . . . . . . . . . . . . . . 78
4.3.3 Copper Oxide as Enzyme Mimics . . . . . . . . . . . . . . . . . . . . 81
4.3.4 MoO3, TiO2, MnO2, RuO2 as Enzyme Mimics . . . . . . . . . . 81
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
5 Other Nanomaterials for Nanozymes . . . . . . . . . . . . . . . . . . . . . . . . . . 93
5.1 Prussian Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
5.2 Metal-Organic Frameworks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
5.3 Metal Chalcogenides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
5.4 Metal Hydroxides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
5.5 Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
6 Challenges and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Abbreviations
4-AAP 4-aminoantipyrine
ABTS 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
AgNP Silver nanoparticle
AuNC Gold nanocluster
AuNP Gold nanoparticle
BA Benzoic acid
BSA Bovine serum albumin
CEA Carcinoembryonic antigen
CNT Carbon nanotube
Color. Colorimetric
DAB Diazoaminobenzene
DOPA Dopamine
DPD N,N-diethyl-p-phenylenediamine sulfate
dsDNA Double-stranded DNA
E-chem Electrochemical
ELISA Enzyme-linked immunosorbent assay
EPR Electron paramagnetic resonance
Fluor. Fluorometric
HPNP 2-hydroxypropyl-4-nitrophenylphosphate
HRP Horseradish peroxidase
LDH Layered double hydroxide
LOD Limit of detection
Meth Methods
MNPs Magnetic nanoparticles
NMDA N-methyl-D-aspartate
NPs Nanoparticles
OPD o-phenylenediamine
PDDA Poly(diallyldimethylammonium chloride)
PLGA Poly(D,L-lactic-co-glycolic acid)
PMIDA N-(phosphonomethyl)iminodiacetic acid
ix
x Abbreviations
PSA Prostate-specific antigen

PSS Poly(styrenesulfonate)
PVDF Polyvinylidene difluoride
Ref References
SBA-15 Santa Barbara Amorphous type material
SOD Superoxide dismutase
ssDNA Single-stranded DNA
TMB 3,3′,5,5′-tetramethylbenzidine
Chapter 1
Introduction to Nanozymes
Abstract Natural enzymes play vital roles in biological reactions in living systems.
However, some intrinsic drawbacks, such as ease of denaturation, laborious
preparation, high cost, and difficulty of recycling, have limited their practical
applications. To tackle these problems, intensive efforts have been devoted to
developing natural enzymes’ alternatives called “artificial enzymes.” As an
emerging research area of artificial enzymes, nanozymes, the catalytic nanomate-
rials with enzyme-like characteristics, have attracted researchers’ enormous atten-
tions. In this chapter, after the brief description of the history of nanozymes research
in the course of natural enzymes and artificial enzymes research, a comparison
between nanozymes and natural enzymes as well as artificial enzymes is made.
Such a comparison highlights the unique characteristics of nanozymes, such as their
size-(shape-, structure-, composition-)tunable catalytic activities, large surface area
for modification and bioconjugation, multiple functions besides catalysis, smart
response to external stimuli, etc.

Keywords Natural enzymes Artificial enzymes Nanozymes Enzyme mim-
ics Catalytic nanomaterials
Nanobiology
Functional nanomaterials

Biological catalysts Biomimetic chemistry Nanomaterials with enzyme-like
characteristics
Natural enzymes are ubiquitous biocatalysts that play central roles in virtually all
the biological reactions in living systems [1]. Since they catalyze the reactions with
remarkable efficiency and extraordinary specificity at mild conditions (such as room
temperature, ambient pressure, aqueous solutions, etc.), natural enzymes have been
extensively explored for various applications beyond living systems. For instance,
they have been widely used in biomedicine, clinic, environmental and food
industry. On the other hand, natural enzymes are proteins or ribonucleic acids,
which inevitably have several intrinsic drawbacks, such as ease of denaturation,
laborious preparation, high cost, difficulty of recycling, etc. These drawbacks have
in turn limited their practical applications.
© The Author(s) 2016 1

X. Wang et al., Nanozymes: Next Wave of Artificial Enzymes,
SpringerBriefs in Molecular Science, DOI 10.1007/978-3-662-53068-9_1
2 1 Introduction to Nanozymes
Fig. 1.1 A brief timeline for the development of artificial enzymes (natural enzymes are also
listed for comparison). Reprinted from Ref. [2], Copyright 2016, with permission from Royal
Society of Chemistry
To tackle these drawbacks, intensive efforts have been devoted to developing

natural enzymes’ alternatives called “artificial enzymes” (or “enzyme mimics”) since
1950s (Fig. 1.1) [3]. Artificial enzymes aim at “imitating the catalytic processes
that occur in living systems,” as defined by Breslow [3]. In their pioneering work,
Breslow and others have used cyclodextrins and their derivatives to mimic varieties
of enzymes, ranging from thiamine pyrophosphate and pyridoxal phosphate to
hydrolytic enzymes and even cytochrome P-450 [3]. Inspired by the success of
these studies, researchers have investigated numerous types of materials like metal
complexes, polymers, supramolecules, and biomolecules (such as nucleic acids,
catalytic antibodies, and proteins) for mimicking various kinds of natural enzymes
[3]. For example, synthetic polymers with enzyme-like catalytic activities have
been studied by Klotz et al. [4]. To date, enormous progress has been made in the
field of artificial enzymes (Fig. 1.1), as evidenced by the publication of numerous
excellent reviews and even several monographs on the topic [3, 5–22].
Over the past two decades, along with the remarkable achievements made in the
field of nanotechnology, varieties of functional nanomaterials have been discovered
to possess unexpected enzyme-mimicking catalytic activities (Fig. 1.1). These
emerging functional nanomaterials are now collectively termed as “nanozymes”.
The term “nanozymes” was coined by Pasquato, Scrimin, and their coworkers in
2004 to describe the gold nanoparticle-based transphosphorylation mimics resulting
from the self-assembly of triazacyclonane-functionalized thiols onto the surface of
gold nanoparticles [23]. Later, in their comprehensive review published in 2013,
Wei and Wang defined “nanozymes” as “nanomaterials with enzyme-like charac-
teristics” [24].
As a new type of promising artificial enzymes, nanozymes have attracted con-
siderable attentions, particularly in recent years. As shown in Fig. 1.2, since the
seminal work on fullerene derivatives-based DNase mimics (i.e., their capability for
1 Introduction to Nanozymes 3
Fig. 1.2 Number of published papers on nanozymes by the end of 2015. The data is based on
Web of Science (April 2016)
oxidative DNA cleavage) in the early 1990s [26], incredible growth has been
witnessed in the field of nanozymes by the exponential number of publications. By
the end of 2015, more than 740 papers on nanozymes have been published, among
which 722 were published after 2007. Recently, a special issue has been devoted to
nanozymes [27]. The growing interests in nanozymes can be attributed to their
unique characteristics over natural enzymes and even conventional artificial
enzymes. Nanozymes are unique in several aspects, such as their size- (shape-,
structure-, composition-)tunable catalytic activities, large surface area for modifi-
cation and bioconjugation, multiple functions besides catalysis, smart response to
external stimuli, etc. (Table 1.1) [24]. To date, plenty of nanomaterials have been
investigated to mimic diverse natural enzymes, which have already found many
interesting applications [2, 24, 25, 28–42].
In the following chapters (Chaps. 2–5), nanozymes are discussed based on the
nanomaterials rather than the natural enzymes which they mimic because many
nanomaterials have exhibited multiple enzyme-mimicking activities. We do not
attempt to cover all the published papers on nanozymes in this book. Instead, for
each typical nanomaterial, we mainly focus on its enzyme-mimicking activities,
catalytic mechanisms, and the key applications. In the last chapter (Chap. 6), we
discuss the current challenges and future prospects that nanozyme research is
currently facing to fulfill its great potentials.
Table 1.1 Comparison between nanozymes and othersa, b
Characteristics
Nanozymes (1)Low cost
(2)Easy for mass production
(3)Robustness to harsh environments
(4)High stability
(5)Long-term storage
(6)Tunable activity
(7)Size- (shape-, structure-, composition-) dependent
properties
(8) Multifunction
(9) Easy for further modification (such as bioconjugation)
(10) Smart response to external stimuli
(11) Self-assembly
Conventional artificial (1) Low cost
enzymes (2) Easy mass production
(3) Robustness to harsh environments
(4) High stability
(5) Long-term storage
(6) Tunable activity
(7) Established methods for preparation and characterization
(8) Uniform size and defined structures of molecular mimics
(9) Smaller size (compared with nanozymes)
Natural enzymes (1) High catalytic efficiency
(2) High substrate specificity
(3) High (enantio)selectivity
(4) Sophisticated three-dimensional structures
(5) Wide range of catalytic reactions
(6) Tunable activity
(7) Good biocompatibility
(8) Rational design via protein engineering and computation
a
The items in italic font are unique for nanozymes compared with conventional artificial enzymes
b
The table was adapted from Ref. [24], Copyright 2013, with permission from Royal Society of
Chemistry
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Chapter 2
Carbon-Based Nanomaterials
for Nanozymes
Abstract Carbon-based nanomaterials, such as fullerene, graphene, carbon nan-

otubes, and their derivatives, have been extensively studied to mimic various nat-
ural enzymes owing to their fascinating catalytic activities. In this chapter, their
enzyme mimetic activities (such as nuclease mimics, superoxide dismutase mimics,
peroxidase mimics, etc.) are discussed. The catalytic mechanisms are also discussed
if they have been elucidated. Representative examples for applications, from
biosensing to therapeutics, are covered.

Keywords Nanozymes Artificial enzymes Enzyme mimics Carbon-based

nanomaterials Fullerene and derivatives Graphene and derivatives Carbon

nanotubes Peroxidase mimics Superoxide dismutase mimics Nuclease mimics
Carbon-based nanomaterials, such as fullerene, carbon nanotubes (CNTs), gra-

phene, and their derivatives, have found broad applications in many areas. Owing to
their interesting catalytic activities, they have been extensively studied to mimic
various natural enzymes. In this chapter, we will discuss the nanozymes based on
these carbon nanomaterials.
2.1 Fullerene and Derivatives
Fullerene and its derivatives were among the first nanomaterials that have been
explored for mimicking natural enzymes [1, 2]. In the early 1990s, the light-induced
oxidative DNA cleavage with a C60 derivative (i.e., C60-1) was studied, indicating
that fullerene could mimic natural nuclease [1]. A few years later, the superoxide
dismutase (SOD) mimicking activity of derivatized C60 was investigated, which led
to the long-lasting research interests in fullerene-based nanozymes till today.
Fullerene and its derivatives have also been used to mimic other enzymes besides
nuclease and SOD.

8 2 Carbon-Based Nanomaterials for Nanozymes
2.1.1 Fullerene and Derivatives as Nuclease Mimics
Nuclease catalyzes the cleavage of phosphodiester bond between two nucleotides in

a nucleic acid. Pristine fullerenes including C60 are not water soluble, which makes
them impossible to mimic enzymes in aqueous solution. Therefore, fullerenes have
been solubilized by introducing hydrophilic moieties. Nakamura group have made
water-soluble C60-1 and studied its photoinduced biochemical activities (Fig. 2.1)
[1]. Interestingly, they established that the fullerene carboxylic acid (i.e., C60-1)
oxidatively cleaved DNA under light irradiation. Since C60-1 did not bind to the
DNA to be cleaved, the cleavage was random. To address this issue, Hélène,
Nakamura and coworkers synthesized C60-2, which had a 14-mer DNA sequence
Fig. 2.1 Fullerene derivatives as nuclease mimics. a Light-induced cleavage of DNA with the
fullerene derivative C60-1. b Selective cleavage of DNA by forming a triplex with the fullerene
DNA conjugate C60-2. Adapted from Ref. [2], Copyright 2003, with permission from American
Chemical Society
2.1 Fullerene and Derivatives 9
complementary to the target DNA (Fig. 2.1) [3]. By forming a triplex, the selective
cleavage at guanine-rich sites was achieved. There are other ways to make the
selective cleavage possible. For instance, by conjugating fullerenes with DNA
intercalators (such as acridine), the formed fullerene derivative could enhance DNA
cleavage activity compared with the parent fullerene [4]. These early studies have
established that water-soluble fullerene derivatives could mimic nuclease.
2.1.2 Fullerene and Derivatives as SOD Mimics
(a) Fullerenes as SOD mimics: in vitro activities and mechanisms

Reactive oxygen species (ROS) plays both beneficial and harmful roles in living
systems. Superoxide anion, one of ROS, could cause tissue injury and associated
inflammation if it were not properly regulated. In nature, SOD has been evolved to
catalyze the disputation of superoxide anions into hydrogen peroxide and molecular
oxygen and thus protect living systems from the superoxide anion-induced damage.
To overcome the limits of natural SOD (such as limited stability and high cost),
great efforts have been devoted to developing SOD mimics. The SOD-mimicking
activities of fullerenes have been established by the seminal work from Choi and
coworkers and have since been extensively studied [5].
Inspired by the early discovery that C60 could act as a radical sponge, Choi et al.
studied the neuroprotective activities of two polyhydroxylated C60 (i.e., C60(OH)12
and C60(OH)nOm, n = 18–20, m = 3–7 hemiketal groups) [5, 7]. Surprisingly, both
of the two fullerene derivatives could scavenge free radicals and thus reduce
excitotoxic and apoptotic death of cultured cortical neurons. Later, they identified
that C60[C(COOH)2]3 with C3 symmetry (C60-C3) was more effective toward
neuron protection [8]. Since superoxide anion could be eliminated via either a
stoichiometric scavenging mechanism or a SOD-like catalytic mechanism, sys-
tematic studies including electron paramagnetic resonance (EPR) were carried out
to confirm the SOD-mimicking activity of C60-C3 [6]. The possible stoichiometric
scavenging mechanism was ruled out due to the following facts: first, no structural
modifications to C60-C3 were observed; second, the production of oxygen and
hydrogen peroxide was detected; and the absence of EPR active (paramagnetic)
products. By combining the experimental results with computational data, a cat-
alytic mechanism for SOD-like activity of C60-C3 was proposed (Fig. 2.2). The
proposed mechanism was supported by other studies using dendritic C60 derivatives
and other computational studies [9, 10].
The neuroprotective effects of fullerene-based SOD mimics were also studied
using several other cell lines. For instance, methionine-modified C60 could protect
SH-SY5Y neuroblastoma cells from lead ions (Pb2+) induced oxidative damage and
thus improved the cell survival [11].
Though water soluble fullerenes are usually required for enzyme-mimicking
studies, it has been demonstrated that pristine C60 aqueous suspension was not only
Fig. 2.2 Derivatized C60 as SOD mimics. a C60-C3 as a SOD mimic for catalytically converting
superoxide anion into hydrogen peroxide, water, and hydroxyl. b The proposed catalytic
mechanism. Adapted from Ref. [6], Copyright 2004, with permission from Elsevier
biocompatible but also showed protective effects on liver injury [12]. Besides
derivatization, fullerenes could also be solubilized by using the similar strategies for
hydrophobic drug solubilization. For instance, pristine C60 has been solubilized in
olive oil for various applications [13].
2.1 Fullerene and Derivatives 11
(b) Fullerenes as SOD mimics: in vivo applications

To conclusively demonstrate that C60-C3 could work in living system as a SOD
mimic, Dugan et al. employed SOD2 knockout mice as an in vivo model to
investigate the therapeutic efficacy of C60-C3 (Fig. 2.3) [6]. The SOD2 knockout
mice would die in utero or within a few days after birth owing to mitochondria
damage by oxidative species. Therefore, they were suitable models to evaluate the
in vivo effects of SOD mimics. It showed that the life span of the SOD2 defective
mice could be extended by 300 % when C60-C3 was administrated both in utero
and postnatally, demonstrating the C60-C3 could be functional alternatives to SOD2
in the studied mice. SOD2 is a manganese SOD localized in the mitochondria.
Further immunostaining indeed revealed that C60-C3 was uptaken and localized to
mitochondria [6].
These results indicated that fullerenes with SOD-mimicking activities (such as
C60-C3) may hold translational promise for treating several diseases in future.
Fig. 2.3 a Treatment of SOD2 knockout mice with C60-C3. b Percentage of Sod2−/− pups born to
Sod2+/− parents. Pregnant dams were given C60-C3 in their drinking water starting at Day 14–15 of
pregnancy. Control dams received dilute red food coloring. The percentages of Sod2−/− pups, per
litter, born to control versus C60-C3-treated dams were, respectively, 6 ± 2 % (controls, 11 L) and
20 % ± 2 (C3, 9 L) (mean ± standard error of mean, with p = 0.03 by t test, and p = 0.04 by the
nonparametric Wilcoxon rank sum test; the theoretical expected percentage is 25 %), indicating in
utero rescue of some Sod2−/− embryos. c Survival (days) of Sod2−/− pups treated with daily
subcutaneous injection of C60-C3 or color-matched food coloring until death. Values are
means ± standard error of mean, *p < 0.05 by t test, n = 9, C60-C3 versus n = 7, vehicle. Adapted
from Ref. [6], Copyright 2004, with permission from Elsevier
2.1.3 Fullerene Derivatives as Peroxidase Mimics
A few studies also showed that fullerenes could mimic peroxidase [14, 15]. For
instance, the peroxidase-mimicking activity of C60[C(COOH)2]2 was demonstrated
by its catalyzed oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) with H2O2 [15].
2.2 Graphene and Derivatives
Peroxidase catalyzes the oxidation of its substrates with H2O2 into oxidized
products. The products are usually either colored or fluorescent, which enables
peroxidase for a wide range of bioanalytical and biomedical applications. The
peroxidase-mimicking activities of nanomaterials were initially studied with Fe3O4
nanoparticles by Yan and coworkers in 2007 [16]. Inspired by Yan’s work, Wang
et al. developed a general sensing strategy using Fe3O4 nanoparticles-based per-
oxidase mimic in 2008 (see detailed discussion in Chap. 4) [17]. Since then, lots of
different nanomaterials have been investigated to mimic peroxidase. Among them,
graphene and its various derivatives have showed great promise in mimicking
peroxidase.
Graphene and its derivatives with peroxidase-mimicking activities can be
roughly classified into two types. For the first type, the activities are solely from
graphene or its derivatives. For the second type, the activities are either from the
catalysts assembled onto graphene (or its derivatives) or from the synergistic effects
of both the decorated catalysts and graphene (or its derivatives). Note: other
enzyme-mimicking activities of graphene and its derivatives still remain to be
investigated [18].
2.2.1 Graphene and Its Derivatives as Peroxidase Mimics
The peroxidase-mimicking activities have been studied mainly with graphene

derivatives since pure graphene without any modifications is not water soluble. Qu
and coworkers reported the intrinsic peroxidase-mimicking activity of graphene
oxide with carboxyl modifications (GO-COOH) [19]. The activity was first
demonstrated by the catalytic oxidation of TMB with H2O2 in the presence of
GO-COOH (Fig. 2.4). The kinetic studies revealed that GO-COOH had higher
affinity toward TMB in comparison with natural peroxidase. Interestingly, the
GO-COOH catalyzed reactions proceeded via a ping-pong mechanism, which was
the same as that for natural peroxidase. Since they did not detect trace amount of
metal catalysts, they attributed the observed catalytic activity to the GO-COOH
itself. No mechanism responsible for the catalytic activity was proposed, though it
suggested that electron transfer from GC-COOH to H2O2 may be involved. By
2.2 Graphene and Derivatives 13
Fig. 2.4 Carboxyl-modified graphene oxide as peroxidase mimic. a Typical photographs of the
reaction solutions incubated at room temperature in pH 5.0 phosphate buffer (from left to right):
(1) 50 mM H2O2 and 800 μM TMB, colorless; (2) 40 mg/mL GO-COOH, black; (3) 50 mM
H2O2, 800 mM TMB and 40 mg mL/mL GO-COOH, turning blue. b The time-dependent
absorbance changes at 652 nm in the absence (black) or presence of different concentrations of
GO-COOH in pH 5.0 phosphate buffer at room temperature. Reprinted from Ref. [19], Copyright
2010, with permission from John Wiley and Sons
combining with natural glucose oxidase (GOx), the glucose in diluted blood and
fruit juice samples was successfully detected using the GC-COOH-based peroxi-
dase mimic [19].
Graphene derivatives and many other carbon-based nanomaterials have rich
oxygenated functional moieties (such as hydroxyl, ketone, carboxyl, epoxide, etc.).
These functional moieties may play key roles in their enzyme-mimicking activities.
To figure out the possible functional moieties responsible for the peroxidase-
mimicking activity of a graphene derivative called graphene quantum dots (GQDs),
several reagents were employed to selectively deactivate these functional moieties
(Fig. 2.5) [20]. GQDs are small pieces of graphene derivative with fluorescent
properties. It has ketone, hydroxyl, and carboxyl groups on the surface. These three
groups can selectively react with phenylhydrazine (PH), benzoic anhydride (BA),
and 2-bromo-1-phenylethanone (BrPE) respectively (Fig. 2.5). After treatment with
PH, BA, BrPE, the peroxidase-mimicking activities of the treated GQDs were
studied. As shown in Fig. 2.5b, the activity of PH-treated GQDs was significantly
inhibited while the activity of BA-treated GQDs was enhanced. The activity of
BrPE-treated GQDs remained almost the same as the untreated GQDs. Using a HO∙
specific fluorescent probe, the formation of HO∙ was confirmed during the catalytic
reaction. Further kinetic measurements and theoretical calculation were also carried
out. Taking together, these results suggested that ketone groups were the catalyti-
cally active sites and the carboxyl groups were the substrate binding sites. The
hydroxyl groups, on the other hand, played an inhibitory role. It may be applicable
to other carbon nanomaterials-based peroxidase mimics, which also have such
oxygenated moieties.
Fig. 2.5 Deciphering

peroxidase-mimicking
activity of GQDs. a Reactions
involved in selectively
deactivating functional
moieties on GQDs. b Relative
catalytic activities of GQDs
treated with different reagents.
Adapted from Ref. [20],
Copyright 2015, with
permission from John Wiley
and Sons
Since HO∙ was involved in peroxidase-mimicking activity of GQDs, they have

showed antibacterial activity even in the low level of H2O2. Both Gram-positive
(Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria could be
inhibited with the GQDs. The in vivo antibacterial efficacy was then evaluated
using a mouse injury model, showing that the combination of GQDs with H2O2
exhibited the best therapeutic effects compared with saline, H2O2, and GQDs [21].
The water solubility and stability of GO could be further enhanced by coating
with polymers. To this end, chitosan, a cationic polysaccharide, was used to coat
GO. The obtained chitosan–GO showed improved stability toward catalytic oxi-
dation of TMB with H2O2. Interestingly, it was found that the peroxidase-
mimicking activity of the chitosan–GO was regulated by light [22]. Under visible
light irradiation, the catalytic activity was turned on. More, the coated chitosan
could interact with concanavalin A (Con A) via a multivalent manner, and the
interaction would induce the aggregation of chitosan–GO. Such aggregation in turn
reduced the activity of the nanozyme. Glucose, however, could compete for the
binding sites of chitosan on Con A due to the stronger interaction between glucose
and Con A. Therefore, the presence of glucose would disassemble the chitosan–
GO/Con A aggregates and thus recover the catalytic activity. Based on this phe-
nomenon, a facile phototriggered method for glucose detection was developed.
A linear range of 2.5–5.0 mM for glucose was achieved [22].
2.2.2 Decorated Graphene (or Its Derivatives) as Peroxidase

Mimics
The large surface area of graphene and its derivatives provides a good opportunity
to decorate them with various functional molecules and nanomaterials. As men-
tioned above, for such decorated graphene (or its derivatives), the peroxidase-
mimicking activities could be originated from either the assembled catalyst itself or
the catalyst/graphene (or its derivatives) assembles.
In their seminal report, Dong and coworkers assembled hemin onto reduced GO
(denoted as rGO) to form hemin–rGO complex and studied its peroxidase-
mimicking activity (Fig. 2.6). The rGO was obtained by reducing GO with
hydrazine. Then the hemin–rGO was prepared through the π-π stacking interaction
between hemin and rGO. Compared with hemin–rGO, rGO showed almost negli-
gible activity. Therefore, the peroxidase-mimicking activity of the hemin–rGO was
mainly attributed to the assembled hemin. As shown in Fig. 2.6, the hemin–
rGO-based nanozyme could catalyze the oxidation of TMB, ABTS (2,2′-azinobis
(3-ethylbenzothiozoline)-6-sulfonic acid, and OPD (o-phenylenediamine) into the
corresponding colored products with H2O2. Kinetic studies revealed that the
hemin–rGO catalyzed reaction also followed a ping-pong mechanism [23].
Fig. 2.6 Hemin-decorated rGO as peroxidase mimic. a Schematic illustration of peroxidase-like

activity of hemin–rGO. b Hemin–rGO catalyzed oxidation of various peroxidase substrates with
H2O2 to the corresponding colored products. Adapted from Ref. [23], Copyright 2011, with
permission from American Chemical Society
It is known that single-stranded DNA (ssDNA) and double-stranded DNA

(dsDNA) have different affinity toward various nanomaterials [24]. ssDNA binds
tightly onto graphene (or its derivatives) while dsDNA binds weakly. Such a dif-
ference could be further amplified by salt-induced aggregation. In the presence of
high concentration of salt (such as NaCl), ssDNA protects graphene (or its
derivatives) from aggregation while dsDNA cannot. Based on this phenomenon,
Dong et al. went on further to develop a label-free colorimetric method for
single-nucleotide polymorphisms (SNPs) (Fig. 2.7) [23]. The probe ssDNA could
stabilize the hemin–rGO and thus retained its peroxidase-mimicking activity. When
the complementary target ssDNA was hybridized with the probe ssDNA, the
formed dsDNA could not stabilize the hemin–rGO, which led to significant inhi-
bition of its peroxidase-mimicking activity and produced the weakest signal. When
a target ssDNA with a single-base mismatch was introduced, the formed duplex
could partially protect the hemin–rGO from aggregation and thus retained part of its
peroxidase-mimicking activity. This in turn resulted in the signal with medium
intensity. As shown in Fig. 2.7, one could easily distinguish the single-base mis-
matched target DNA from the complementary one even with naked eyes [23].
This sensing strategy could be applicable to functional nucleic acids (such as
aptamers) [24, 25]. An aptamer is an ssDNA or ssRNA that can specifically bind to
its target. The binding usually induces a conformational change of the aptamer.
Such a conformational change could in principle be sensed with hemin–rGO. For
Fig. 2.7 Hemin-decorated rGO as peroxidase mimic for single-nucleotide polymorphisms.

a Protocol for SNPs detection. (a) Probe ssDNA (no precipitation, dark blue), (b) single-base
mismatched duplex DNA (small amount of precipitation, blue), and (c) complementary duplex
DNA (much precipitation, light blue). b Time-dependent absorbance changes in the presence of
different amounts of target ssDNA. c Time-dependent absorbance changes with corresponding
supernatant in (a) ssDNA, (b, c, d) single-base mismatched duplex DNA, and (e) complementary
duplex DNA. Reprinted from Ref. [23], Copyright 2011, with permission from American
Chemical Society
instance, when an aptamer for acetamiprid (an insecticide) was used to stabilize
hemin–rGO, the nanozyme showed high activity. However, the presence of acet-
amiprid would form the acetamiprid-aptamer complex, which did not protect the
hemin–rGO efficiently. Therefore, the nanozyme’s activity was significantly
inhibited due to the aggregation. With this sensing strategy, as low as 40 nM
acetamiprid was detected [26]. Other targets of interests can also be detected when
the corresponding aptamers are used.
The synergistic effects were observed for numerous decorated graphene (or
its derivatives) [27–35]. Among GO, rGO, AuNPs, the mixture of rGO and
AuNPs, and the gold nanoparticles-decorated rGO (denoted as AuNPs@rGO),
AuNPs@rGO exhibited the highest peroxidase-mimicking activity. As shown in
Fig. 2.8, the significantly enhanced catalytic activity of the AuNPs@rGO was
attributed to the synergistic effects. Careful study suggested that the synergistic
effects were due to: (a) the strong interaction between Au 5d of AuNPs and C 2p of
Fig. 2.8 AuNPs@rGO with synergistic peroxidase-mimicking activity and its use for DNA
sensing. a TEM (transmission electron microscopy) image of AuNPs@rGO.
b Peroxidase-mimicking activities of various nanomaterials. c Comparison of fluorescent retention
ratio of FAM-labeled probe ssDNA and the corresponding dsDNA after incubated with
AuNPs@rGO. d Time-dependent absorbance changes with varying concentrations of target
ssDNA. Adapted from Ref. [27], Copyright 2012, with permission from American Chemical
Society
rGO at the interface, which was favorable to H2O2 and HO∙ absorption; and (b) the
modified electronic structure and Fermi level of rGO owing to the above-mentioned
interfacing, which in turn resulted in the n-type doping of rGO and accelerated the
catalytic reactions [27]. More, by further exploring the different affinities of ssDNA
and dsDNA toward the AuNPs@rGO, a general analytical strategy for target DNA
was developed (Fig. 2.8). The ssDNA specific nuclease (such as S1 nuclease) was
also successfully detected using the developed method, where the presence of S1
nuclease would cleave the probe ssDNA into small fragments and thus recover the
nanozyme’s activity. If an aptamer was used as the probe ssDNA, the corre-
sponding target (such as insulin) could be detected [27].
A hybrid called GSF@AuNPs with peroxidase-mimicking activity was prepared
by in situ formation of AuNPs onto sandwich-like mesoporous silica/rGO. The
silica/rGO was pre-conjugated with folic acid for tumor cell recognition [29]. The
nanozyme was then used for detection of cancer cells with overexpressed folate
receptor. For instance, rapid detection of HeLa cells was achieved. More, since HO∙
was generated during the catalysis, the nanozyme was also used to selectively kill
cancer cells with the help of either exogenous or endogenous H2O2 [29].
Chen and coworkers prepared PtNPs decorated GO (i.e., PtNPs/GO) and further
modified it with folic acid [36]. Based on the peroxidase-mimicking activity of the
folic acid-PtNPs/GO, a colorimetric assay for cancer cells was developed (Fig. 2.9).
With the developed assay, as few as 125 cancer cells have been detected by naked
eyes.
By conjugating the monoclonal antibody for aflatoxin B1 with a
peroxidase-mimicking nanozyme, an electrochemical immunoassay for aflatoxin B1
was reported by Tang et al. [32]. The nanozyme had a structure of
PtNPs/CoTPP/rGO, in which CoTPP was 5,10,15,20-tetraphenyl-21H,23H-
porphine cobalt. Aflatoxin B1 is a highly toxic metabolite secreted by food fungi
and its detection still needs rapid methods with high sensitivity and low cost. With
Fig. 2.9 Colorimetric detection of cancer cells with folic acid–PtNPs/GO as a peroxidase mimic.
Reprinted from Ref. [36], Copyright 2014, with permission from American Chemical Society
the developed immunoassay, as low as 5.0 pg/mL of aflatoxin B1 was successfully

detected. Besides, the immunoassay has been used for analyzing real samples, such
as the naturally contaminated peanut samples, showing good agreement with
ELISA (enzyme-linked immunosorbent assay) kit [32].
Yu and coworkers reported a disposable electrochemical immunosensor based
on peroxidase-mimicking nanozyme for cancer antigen 153 (CA153) detection
[34]. Their nanozyme had a structure of ZnFe2O4@silica/GO (Fig. 2.10). The
antibodies were conjugated onto the silica shell. Using a sandwich assay format,
sensitive and selective detection of CA153 was achieved with a dynamic range
from 10−3 to 200 U/mL and a detection limit of 2.8 × 10−4 U/mL. The fabricated
immunosensor was further used to detect CA153 in serum samples and the results
were consistent with the clinical ones.
Fig. 2.10 Peroxidase-mimicking nanozyme and its use for disposable electrochemical
immunosensor. Reprinted from Ref. [34], Copyright 2014, with permission from Elsevier
2.3 Carbon Nanotubes
CNTs as well as decorated CNTs have been used to mainly mimic peroxidase
though other CNT-based enzyme mimics were also reported [37, 38].
2.3.1 Carbon Nanotubes as Peroxidase Mimics
Qu and coworkers reported the peroxidase-mimicking activity of single-walled

carbon nanotubes (SWNTs) [39]. The activity of SWNTs was investigated by
catalytic oxidation of TMB with H2O2. Since metal catalysts are usually used to
grow SWNTs, a trace amount of metal catalyst residues rather than SWNTs
themselves may be responsible for the mimicking activity. To address this concern,
the sonication-assisted washing with mixed acids (i.e., a mixture of concentrated
sulfuric and nitric acids) was carried out to completely remove the metal residues
(i.e., Co). It was found that pristine SWNTs and treated SWNTs did not show any
significant differences in their catalytic activities. This confirmed that the
peroxidase-mimicking activity of SWNTs was from the SWNTs themselves instead
of the metal residues. By exploring the different affinities of ssDNA and dsDNA
toward SWNTs, they developed a colorimetric assay for DNA detection [39].
It should be noted that in the above study, only the effect of Co on SWNTs’
peroxidase-mimicking activity was tested. Other metal residues may have different
effects. Zhu and coworkers indeed found that Fe content in the helical CNTs played
an important role in their catalytic activities [40]. As shown in Fig. 2.11, the more
Fe content of helical CNT was, the higher its peroxidase-mimicking activity of
helical CNT was. Even for the helical CNT with the lowest amount of Fe, its
activity was still higher than that of MWNTs (multiwalled CNTs). Zhu’s and Qu’s
results suggest that more systematic studies are needed to decipher the exact
mechanisms of CNTs’ enzyme-mimicking activities. Zhu et al. then fabricated an
electrochemical sensor using the helical CNTs as peroxidase mimic for H2O2
detection [40].
Like graphene, the decoration of CNTs could also synergistically enhance their
peroxidase-mimicking activities. When MWNTs were decorated with magnetic
silica nanoparticles, the decorated MWNTs exhibited higher peroxidase-mimicking
activity compared with the individual components (i.e., MWNTs and magnetic
silica nanoparticles, respectively) [41]. Since the decoration of magnetic silica
nanoparticles onto MWNTs was achieved by the Cu2+-mediated click chemistry, a
colorimetric assay for Cu2+ was proposed (Fig. 2.12). The decorated MWNTs
could be concentrated by a magnet and then used to oxidize TMB to its colored
products. On the other hand, the undecorated MWNTs would be washed away and
only the magnetic silica nanoparticles would be concentrated by a magnet. The
latter exhibited much lower catalytic activity compared with the decorated
2.3 Carbon Nanotubes 21
Fig. 2.11 Peroxidase-mimicking activity of helical CNTs. a SEM (scanning electron microscopy)
image of helical CNTs. b Peroxidase-mimicking activities of helical CNTs with different Fe
contents as well as MWNTs. The catalytic reaction without CNTs was shown as a control.
Adapted from Ref. [40], Copyright 2011, with permission from John Wiley and Sons
MWNTs. Via such a sensing strategy, high sensitive and selective detection of Cu2+
has been carried out [41].
Other decorated CNTs with synergistically enhanced peroxidase-mimicking
activities have also been developed and used to detect biologically important targets
Fig. 2.12 Cu2+ detection using magnetic silica nanoparticles clicked on MWNTs. a The sensing
mechanism. b The time-dependent absorbance changes in the absence (black) or presence of
different concentration of Cu2+. c Calibration curve for variable concentrations of Cu2+. The error
bars represent the standard deviation of three measurements. Reprinted from Ref. [41], Copyright
2010, with permission from Royal Society of Chemistry
(also see Tables A1 and A2). For example, when MWNTs were filled with Prussian
blue nanoparticles, the formed nanozyme has been used for colorimetric detection
of H2O2 [42]. When the nanozyme was further combined with glucose oxidase, it
has been successfully used for glucose detection with a linear range of 1 μM to
1.0 mM and a detection limit of 200 nM. The potential practical application was
demonstrated by detecting glucose in serum samples, showing satisfactory recov-
eries of 94–106 % [42]. Cholesterol levels in milk powder were evaluated by using
ZnO nanoparticles-decorated CNTs as a peroxidase mimic [43]. Cholesterol was
catalytically oxidized with cholesterol oxidase to produce H2O2, which was then
used for oxidizing ABTS into the colored product with the nanozyme.
A colorimetric approach to detection of D-alanine was developed by using Au
nanoparticles-decorated SWNTs as a peroxidase mimic [44]. D-alanine was cat-
alytically oxidized with D-amino acids oxidase to produce H2O2 for the further
oxidation of TMB. The proposed approach showed high selectivity and high sen-
sitivity toward D-alanine detection.
A paper-based immunoassay was developed using ZnFe2O4-decorated MWNTs
as a peroxidase mimic [45]. Carcinoembryonic antigen (CEA) was chosen due to its
correlation with cancer. The nanozymes-based immunoassay exhibited high sen-
sitivity and robustness. More, the immunoassay has been used to detect CEA in
clinical serum samples, showing consistent results compared with a commercialized
ELISA method [45].
2.3.2 Carbon Nanotubes as Other Enzyme Mimics
As discussed above, it has been established that fullerenes can efficiently scavenge
radicals due to their SOD-mimicking activities. Thus, it is reasonable to investigate
the radical scavenging activities of CNTs. Tour and coworkers have studied the
radical scavenging activities of several SWNTs (Fig. 2.13) [37]. They used butylated
hydroxytoluene (BHT), a phenolic antioxidant, to modify the SWNTs. The anchored
BHT would endow the SWNTs with radical scavenging activities. Pristine SWNT
(SWNT-1) was not water soluble. Therefore, it was solubilized by either wrapping a
polymer (such as Pluronic for SWNT-2) or introducing carboxyl moieties by mixed
acid treatment/cleavage (i.e., SWNT-3). SWNT-3 was further modified with PEG
(poly(ethylene glycol)) to produce SWNT-4, which was soluble in buffer.
The radical scavenging activities were evaluated by comparing with Trolox, a
vitamin E derivate. The TME (Trolox mass equivalence) values were then deter-
mined. Unexpectedly, SWNT-4 without BHT modification exhibited nearly 40
times higher activity (Fig. 2.14). This indicated that SWNTs alone could act as
antioxidants. For SWNT-5, amine-BHT was assembled via electrostatic interac-
tions while amine-BHT was covalently conjugated for SWNT-6. Since electrostatic
binding was more efficient than the covalent binding, more amine-BHT should bind
onto SWNT-5 than SWNT-6. As expected, SWNT-5 showed higher radical
scavenging activity than SWNT-6 did (Fig. 2.14). For SWNT-2 and SWNT-7, one
2.3 Carbon Nanotubes 23
Fig. 2.13 SWNTs used for scavenging radicals. Adapted from Ref. [37], Copyright 2009, with
would expect that SWNT-7 would have higher activity since it had extra BHT
moieties. Surprisingly, SWNT-7 exhibited lower activity when compared with
SWNT-2 (Fig. 2.14) [37]. This unexpected result indicated that pristine SWNTs
had higher radical scavenging activities than BHT. This study demonstrated that
SWNTs could act as potent antioxidant without acute cellular toxicity.
Fig. 2.14 Radical scavenging activities (i.e., the TME values) of the SWNTs studied. C60-5 was
also included for a comparison. Adapted from Ref. [37], Copyright 2009, with permission from
American Chemical Society
Note that though it has established that fullerenes act as SOD mimic to scavenge
radicals, whether CNTs share a similar SOD mimetic mechanism still remains to be
investigated.
2.4 Other Carbon-Based Nanomaterials
Other carbon-based nanomaterials (such as carbon nanohorn, carbon nanodots,

carbon nanoclusters, etc.) have also been used to mimic peroxidase and other
enzymes [46–58].
2.4.1 Other Carbon Nanomaterials as Peroxidase Mimics
Several groups studied the peroxidase-mimicking activities of carbon nanohorns,

carbon nanodots, etc. [46–48, 50–52, 58, 59]
For instance, Xu and coworkers demonstrated that carboxyl-functionalized
single-walled carbon nanohorns exhibited peroxidase-like activity (Fig. 2.15) [46].
When the nanozyme was further combined with glucose oxidase, a facile colori-
metric assay for glucose was developed. Carbon nanodots with an average size of
2.5 nm also showed peroxidase-mimicking activity and have been used for glucose
sensing (Fig. 2.15c) [51]. The peroxidase-like activity of selenium-doped graphitic
carbon nitride nanosheets was demonstrated and further explored for xanthine
detection when facilitated with xanthine oxidase [52].
Using [Cu3(BTC)2] (BTC = 1,3,5-benzene tricarboxylate) as a precursor, copper
nanoparticles-decorated carbon nanocomposite was fabricated via a one-pot ther-
molysis method (Fig. 2.15d) [47]. It demonstrated that the nanocomposite exhibited
peroxidase mimetic activity. Ascorbic acid, a biologically important antioxidant,
could competitively inhibit the catalytic oxidation of peroxidase substrate (such as
TMB in this study). Based on this inhibition phenomenon, a colorimetric method
for ascorbic acid was developed [47, 60]. The ascorbic acid content in tablets has
been successfully determined with the nanozyme-based method [47].
2.4.2 Other Carbon Nanomaterials as SOD Mimics
The SOD-mimicking activities of nitrogen-doped carbon nanodots have been

studied [58]. It demonstrated that primary amine were the optimal reagents for
nitrogen doping. When the obtained nitrogen-doped carbon nanodots were added to
H2O2-treated cells, they would enhance the cell viability in a concentration-
dependent manner. It suggested that the nitrogen-doped carbon nanotdots protect
cells from H2O2-induced injury by eliminating the ROS and stimulating the native
SOD expression [58].
References 25
Fig. 2.15 Carbon nanomaterials as peroxidase mimics. a Single-walled carbon nanohorns as

peroxidase mimic and b their use for glucose detection. c Carbon nanodots as peroxidase mimics.
d Copper nanoparticles-decorated carbon as peroxidase mimics. a and b Reprinted from Ref. [46],
Copyright 2015, with permission from Royal Society of Chemistry. c Reprinted from Ref. [51],
Copyright 2011, with permission from Royal Society of Chemistry. d Reprinted from Ref. [47],
Copyright 2014, with permission from John Wiley and Sons
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Chapter 3
Metal-Based Nanomaterials for Nanozymes
Abstract The use of metal nanomaterials for mimicking natural enzymes is dis-
cussed in this chapter. These nanozymes are roughly classified into two types: for
type I, the nanozymes’ activities are entirely from the assembled monolayer onto a
metallic core rather than the core itself; for type II, the nanozymes’ activities are
originated from the metal nanomaterials themselves. For both of them, their enzyme
mimetic activities (such as RNase mimics, DNase mimics, superoxide dismutase
mimics, peroxidase mimics, catalase mimics, etc.) are discussed. The catalytic
mechanisms for the multiple enzyme mimicking activities of metal nanomaterials
are elucidated by combing computational studies with experimental results.
Representative examples for applications, from biosensing and immunoassays to
bioimaging and therapeutics, are covered.

Keywords Nanozymes Artificial enzymes Enzyme mimics Metal nanoma-

terials Multiple enzyme mimics Catalytic monolayer-protected gold nanopar-

ticles Immunoassays Self-assembly Computational study Bioanalysis
The metal-based nanozymes could be roughly classified into two types: for type I,
the nanozymes’ activities are entirely from the assembled monolayer onto a metallic
core rather than the core itself; for type II, the nanozymes’ activities are originated
from the metal nanomaterials themselves. In this chapter, both of them are dis-
cussed to highlight their various enzyme mimicking activities and wide
applications.
3.1 Metal Nanomaterials with Catalytic

Monolayers (Type I)
Metal nanomaterials (such as Au, Ag, etc.) with self-assembled monolayers (par-
ticularly the thiolated monolayers) have been extensively explored due to their great
importance to nanotechnology [1]. If catalytic moieties were introduced into the

32 3 Metal-Based Nanomaterials for Nanozymes
monolayers, one would expect the metal nanomaterials protected by the monolayers
to be catalytically active. These functionalized metal nanoparticles could indeed
exert enzyme-like catalysis, and therefore, they have been regarded as nanozymes
[2–5]. These nanozymes could be further classified into three subtypes according to
the molecules used for the monolayers: the first one uses alkanethiol terminated
with catalytic moieties; the second one uses alkanethiol without catalytic terminus,
and the catalytic moieties, instead, are further assembled onto (or into) the mono-
layers; and the third one uses thiolated catalytic biomolecules (such as thiolated
DNAzymes).
3.1.1 AuNPs Protected by Alkanethiol with

Catalytic Terminal Moieties
(a) Alkanethiol-protected AuNPs as RNase mimics: activities and mechanisms

As mentioned in Chap. 1, in their initial study, Scrimin and co-workers used
alkanethiol terminated with catalytic moieties to modify the AuNP core (Fig. 3.1).
The obtained AuNPs assemblies were then used as a metallonuclease (i.e., RNase)
mimic to catalyze the transphosphorylation of 2-hydroxypropyl p-nitrophenyl
phosphate (HPNPP) [2].
Fig. 3.1 Alkanethiol-protected AuNPs as metallonuclease mimics. a Transphosphorylation

catalyzed by RNase A. b Structure of AuNP-1 nanozyme. c Transphosphorylation of HPNPP
catalyzed by AuNP-1 nanozyme. c Adapted from Ref. [2], Copyright 2004, with permission from
John Wiley and Sons
3.1 Metal Nanomaterials with Catalytic Monolayers (Type I) 33
As shown in Fig. 3.2, in comparison with the uncatalyzed transphosphorylation

of HPNPP, the reaction has been accelerated by more than 4 orders of magnitude
when AuNP-1 nanozyme was used. More, even compared with the unassembled
catalytic molecule Zn-1 (i.e., the complex of 1,4,7-triazacyclononane (TACN) and
zinc ion), AuNP-1 nanozyme exhibited more than 600 times rate acceleration. It
should note that the AuNP-1 nanozyme catalyzed reaction also obeyed a typical
Michaelis-Menten kinetics (Fig. 3.2d) [2, 5]. When alkanethiol terminated with
ammonium was used to modify the AuNPs, the formed AuNP-based complexes
were practically inactive. This control experiment confirmed that the catalytic
activity of AuNP-1 nanozyme was from the assembled monolayers rather than the
AuNP core. The AuNP-1 nanozyme could also catalyze the cleavage of RNA
dinucleotides (such as ApA, CpC, and UpU) [2].
Detailed studies revealed that the superior catalytic activity of AuNP-1 nano-
zyme could be attributed to its several unique features [2, 5]. First, it has been
showed that the effective local concentration of guest molecules (e.g., the catalytic
substrate HPNPP in this case) could be significantly enhanced due to both the
electrostatic and hydrophobic interactions between the positively charged
Fig. 3.2 a Comparison of catalyzed and uncatalyzed transphosphorylation of HPNPP. b Structure

of Zn-1 complex. c Proposed catalytic pocket of AuNP-1 nanozyme. d Michaelis-Menten
saturation kinetics of the transphosphorylation catalyzed by AuNP-1. E Dependence of the rate
constant for the transphosphorylation of HPNPP with AuNP-1 on Zn ion concentration. c and
d Adapted from Ref. [5], Copyright 2015, with permission from American Chemical Society.
e Adapted from Ref. [2], Copyright 2004, with permission from John Wiley and Sons
monolayer and the guest molecules [5, 6]. Second, the assembled catalytic moieties
(i.e., the complex of TACN-Zn2+) were multivalent and exhibited cooperative
behavior toward the catalysis. The cooperativity was indicated by the sigmoidal
curve for zinc ion concentration-dependent catalytic activities (Fig. 3.2e) [2, 5]. The
cooperative effect was further validated by experiments and theoretical analysis [7].
It suggested that two neighboring catalytic moieties (i.e., two TACN-Zn2+) were
required to form a catalytic pocket for the cooperative catalysis (Fig. 3.2c) [2, 5, 7].
Such a catalytic pocket mimicked the ones in natural enzymes. Third, a 0.4 unit of
pKa decrease of the assembled TACN-Zn2+ complexes due to the close proximity
effect may also play a role in the enhanced activity [2]. Fourth, the strong Au-S
interaction made the self-assembly very easy to be carried out. Also it endowed the
assembled AuNP-based catalytic complexes with much higher stability compared
with micelle-based systems [5]. Moreover, its catalytic activity could be rationally
regulated. Since the catalytic activity of AuNP-1 nanozyme was from the com-
plexed Zn2+, the activity could be reversibly switched off by using a Zn2+ chelating
reagent and could be subsequently restored by re-adding Zn2+ [8].
For natural metallonucleases, their active sites have low dielectric constant (ε).
As shown in Eq. 3.1 below, the electrostatic interaction between the enzyme and its
substrate would be favored at active sites of low dielectric constant. To mimic such
a unique microenvironment, low polarity should be introduced into the active sites.
Eelectrostatic / ðQ1 Q2 Þ=ðe r1;2 Þ ð3:1Þ
To test this hypothesis, AuNP-based nanozymes with different polarities have

been prepared and their metallonuclease mimicking activities have been studied [9].
As shown in Fig. 3.3, AuNP-3 and AuNP-5 with lower polarities exhibited higher
activities while AuNP-2 and AuNP-4 with higher polarities exhibited lower
activities. Therefore, the catalytic activities were well correlated with the polarities
Fig. 3.3 a AuNP-based nanozymes with different polarities. b Transphosphorylation of HPNPP

with AuNP-based nanozymes, highlighting the dianionic transition state. c Rate of HPNPP
cleavage with different nanozymes. Adapted from Ref. [9], Copyright 2014, with permission from
of the assembled monolayers. The lowered polarities would enhance the electro-
static interaction between the dianionic transition state and the active sites of the
nanozymes (i.e., Zn2+ complex), which therefore improved the catalytic activities.
This study also suggested that catalytic activities of the nanozymes could be tuned
by modulating the monolayers assembled.
(b) Alkanethiol-protected AuNPs as RNase mimics: applications
The above-mentioned RNA mimics have been explored for numerous interesting
applications [10–12]. For instance, colorimetric detection of natural enzyme activity
through catalytic signal amplification with AuNP-1 nanozyme has been reported
[10]. As shown in Fig. 3.4, a natural enzyme substrate (i.e., a peptide in this study)
would interact strongly with AuNP-1 nanozyme and thus inhibit its RNase mim-
icking activity due to the shielding effect. The presence of the natural enzyme
would cleave the substrate into smaller fragments and thus significantly weaken the
interactions with AuNP-1 nanozyme. Therefore, the cleaved fragments would be
released from AuNP-1 nanozyme and thus restore the catalytic activity of AuNP-1
nanozyme. The restored AuNP-1 nanozyme would then convert HPNPP into the
colored product p-nitrophenol, which was used as a reporter. Through the cascade
amplification, the natural enzyme could be determined with higher sensitivity and
selectivity. Moreover, this design was a general approach, which has been extended
to other enzymes by using the corresponding peptide substrates [10].
A sensitive and selective strategy for Hg2+ detection has been demonstrated [11].
AuNP-1 nanozyme had stronger affinity toward a fluorescent reporter than thy-
midine probes (i.e., TDP, TMP, or cTMP), which resulted in the fluorescence
quenching of the reporter. In the presence of Hg2+, the probes would form T-Hg2+-
T complex and the complex could compete against the fluorescent reporter for
binding onto AuNP-1 nanozyme. Therefore, the reporter would be released from
AuNP-1 nanozyme and its fluorescence was recovered. Hg2+ at nanomolar
Fig. 3.4 Detection of enzyme activity through catalytic signal amplification with AuNP-1
nanozyme. Reprinted from Ref. [10], Copyright 2011, with permission from John Wiley and Sons
Fig. 3.5 a Structure of BAPA. b Structure of AuNP-6 nanozyme. c Catalytic cleavage of BNP
with DNase mimic. Adapted from Ref. [13], Copyright 2008, with permission from American
Chemical Society
concentrations has been successfully detected with the proposed sensing strategy. It
has also been demonstrated that AuNP-1 nanozyme could be used to mimic the key
features of cellular signaling pathways [12].
(c) Alkanethiol-protected AuNPs as other enzyme mimics
It is relatively straightforward to design other enzyme mimics by changing the
terminal TACN moiety of the alkanethiol chain to other functional moieties [3, 4,
13–23].
For instance, a DNase mimic has been developed by assembling BAPA termi-
nated alkanethiol onto AuNPs (Fig. 3.5) [13]. The cleavage of BNP (bis-p-nitro-
phenyl phosphate), a DNA model substrate, into MNP (p-nitrophenyl phosphate)
and p-nitrophenolate has been accelerated with AuNP-6 nanozyme by 300,000-
folds over the background cleavage reaction. Moreover, DNA molecules
(such as pBR 322 plasmid DNA) have been successfully cleaved with AuNP-6
nanozyme [13].
DNase could also be mimicked by using AuNPs modified with Ce(IV) complex
terminated alkanethiol monolayers [15]. As many as 2.5 million-fold rate acceler-
ation for the BNP cleavage was observed using the AuNPs-Ce(IV)-based nano-
zymes. The superior catalytic activity was also attributed to the unique features of
the nanozyme, such as the cooperative catalysis.
(d) RNase (DNase) mimics using other supporting cores
Since AuNPs mainly acted as the supporting core, they could be replaced by
other supporting materials. Several studies have showed that by assembling the
catalytic moieties (such as TACN-Zn2+ complex) onto dendrimer, silica, etc.,
RNase and DNase mimics could be obtained (Fig. 3.6) [24–27]. Compared with the
facile Au-S chemistry, the synthesis and purification of these enzyme mimics are
much more difficult and time consuming. Other noble metal nanoparticles (such as
AgNPs), in principle, could also be employed as the supporting core. However, no
study has been reported, which might be due to the easy oxidation of AgNPs.
Fig. 3.6 Dendrimer (a), silica particles (b), and resin (c) as inert supporting materials for catalytic
monolayers. a Adapted from Ref. [24], Copyright 2007, with permission from American Chemical
Society; b Adapted from Ref. [25], Copyright 2009, with permission from Royal Society of
Chemistry; c Adapted from Ref. [26], Copyright 2009, with permission from Elsevier
3.1.2 AuNPs Protected by Alkanethiol with Non-covalently

Assembled Catalytic Moieties
A few studies have showed that the catalytic moieties could be non-covalently
assembled onto (or into) the alkanethiol-protected AuNPs [28–30]. As shown in
Fig. 3.7a, the peptide ligation has been promoted when the two peptide fragments
were electrostatically assembled onto trimethylammonium-functionalized AuNPs
[28]. The transesterification of the p-nitrophenyl ester of N-carboxybenzyl-
phenylalanine was also significantly enhanced by two orders of magnitude when
both the substrate and the catalytic peptide were non-covalently assembled onto
trimethylammonium functionalized AuNPs (Fig. 3.7b) [29]. For both of the
examples, the non-covalent assembly was mainly driven by the electrostatic (and
hydrophobic) interactions. The prominent catalytic activities were owing to the
close proximity of the substrate and the catalyst as well as the unique microenvi-
ronment [28, 29].
Rotello and co-workers developed AuNPs-based nanozymes by encapsulating
hydrophobic molecular catalysts (e.g., Ru complex and Pd complex) within the
hydrophobic region of alkanethiol monolayers on AuNPs cores (Fig. 3.8a) [30]. To
protect the encapsulated molecular catalysts from release, cucurbit[7]uril (CB[7])
was used to cap the head groups of the alkanethiol monolayers. When capped by
CB[7], the nanozymes were inactive. The presence of a competing guest
(i.e., 1-adamantylamine (ADA)) could bind onto CB[7] and form supramolecular
complexes. Therefore, the molecular catalysts were exposed to the surrounding
environment and the nanozymes were subsequently activated. Based on such as
Fig. 3.7 Peptide ligation (a) and transesterification (b) catalyzed by catalysts non-covalently
assembled onto AuNPs. a Reprinted from Ref. [28], Copyright 2007, with permission from
American Chemical Society; b Adapted from Ref. [29], Copyright 2012, with permission from
supramolecular regulation mechanism, the activities of the nanozymes were mod-

ulated in vivo in living cells. As shown in Fig. 3.8, the uptaken nanozymes could
activate nonfluorescent dye for cellular imaging. Moreover, the nanozyme could be
used to activate pro-drug (i.e., pro-5FU) in living cells. With such in situ activation
Fig. 3.8 a Schematic of the nanozyme, consisting of an AuNP core and an alkanethiol monolayer
with encapsulated catalysts. b Cellular uptake of the nanozyme and its regulation. c Activation of
nonfluorescent dye with the nanozyme. d Activation of pro-drug dye with the nanozyme. Adapted
from Ref. [30], Copyright 2015, with permission from Nature Publishing Group
strategy, the toxic side effects of 5FU could be effectively minimized. This study
may provide a facile strategy for tuning the activities of nanozymes in living cells.
3.1.3 AuNPs Protected by Thiolated Biomolecules
Other AuNPs-based nanozymes have been developed by assembling thiolated

biomolecules on the AuNP cores [31–34]. Cao et al. has designed a nanozyme to
mimic the function of RNA-induced silencing complex (RISC) machinery [31].
With the guidance of a regulatory ssRNA, a natural RISC would bind to the
complementary target ssRNA and then silence its function by inducing target RNA
cleavage with a nuclease contained within the RISC. To mimic the features of a
RISC, both nucleases and regulatory ssRNA have been co-assembled onto an
AuNP core (Fig. 3.9a). In the presence of a target ssRNA, the assembled regulatory
ssRNA would form a duplex with the target ssRNA and thus bring it close to the
assembled nuclease for cleavage. To demonstrate the hypothesis, the anti-HCV
(hepatitis C virus) efficacy of the nanozyme was evaluated using a HCV replicon
Fig. 3.9 a Schematic of the nanozyme for RNA silencing. b Anti-HCV effects of the anti-HCV
nanozyme in FL-Neo cells. Adapted from Ref. [31], Copyright 2012, with permission from
National Academy of Sciences
cell culture system (i.e., an FL-Neo cell line). As shown in Fig. 3.9b, the HCV
replication has been successfully inhibited with the nanozyme treatment. The
in vivo efficacy of the nanozyme was then evaluated using a xenotransplantation
mouse model. Remarkably, the nanozyme treatment resulted in more than 99 %
decrease of HCV RNA. Considering the non-detectable cellular interferon response,
the designed nanozyme may be used as a potent nanomedicine for viral infections
and cancers [31]. Other alternative approaches were developed for RNA
interference-independent gene regulation [32, 33, 35, 36].
3.2 Metal Nanomaterials with Intrinsic Enzyme

Mimicking Activities (Type II)
In 2004, the intrinsic oxidase mimicking activity of “naked” citrate-coated AuNPs

was reported [37], since then metal nanomaterials with intrinsic enzyme mimicking
activities have been extensively studied [38–64]. It has been showed that metal
nanomaterials (such as Au, Ag, Pd, Pt, etc.) could mimic oxidase, SOD, catalase,
peroxidase, etc. These nanozymes are unique in several aspects. First, most of them
have multiple enzyme mimicking activities, which are dependent on their
microenvironments. For instance, AuNPs exhibited catalase mimicking activity at
high pH while they showed SOD-like activity at low pH [47]. Second, their enzyme
mimicking activities could be tuned by forming alloys with other metals or by
exposing specific facets [65]. Third, the catalytic activities could also be enhanced
by exploring the plasmonic properties of noble metal nanomaterials [65].
3.2.1 Metal Nanomaterials as GOx Mimics
Glucose oxidase (GOx) catalyzes the oxidation of glucose into gluconic acid
(gluconate) and H2O2 with oxygen. Rossi and co-workers reported the selective
oxidation of glucose with oxygen using AuNPs load on carbon support as the
catalyst [66]. The high selectivity was attributed to the two facts: first, it has
successfully avoided the isomerization of glucose to fructose under acidic condi-
tions; second, it has enhanced the oxidation of the aldehydic group of glucose but
did not affect the oxidation of the alcoholic group [66].
Later they discovered that even citrate-coated AuNPs of 3–6 nm could catalyze
the aerobic oxidation of glucose with dissolved oxygen [37, 39, 40, 67]. The
AuNPs catalyzed reaction was similar to the GOx catalyzed one in terms of the
production of H2O2 and gluconate. Therefore, the AuNPs could be regarded as
GOx mimics. Detailed mechanism studies suggested that the catalytic reaction
followed an Eley–Rideal mechanism (Fig. 3.10) [39, 40, 67]. Briefly, a hydrated
glucose anion first interacted with Au atoms onto the AuNPs’ surface, which would
3.2 Metal Nanomaterials with Intrinsic Enzyme Mimicking Activities (Type II) 41
Fig. 3.10 Proposed molecular mechanism for the GOx-like activity of AuNPs. Reprinted from
Ref. [67], Copyright 2006, with permission from John Wiley and Sons
lead to electron-rich Au species. Then by reacting with the electron-rich Au species

and forming a dioxogold intermediate, oxygen was activated. Two electrons then
transferred from glucose to oxygen via the dioxogold intermediate, producing H2O2
and gluconate [67]. The AuNPs catalyzed reaction also obeyed Michaelis–Menten
kinetics [39, 40]. It also showed that the catalytic activities of the AuNPs were
inversely proportional to their size, further confirming their intrinsic enzyme
mimicking properties [37].
Since Rossi’s work, numerous groups have studied the GOx mimicking activ-
ities of various AuNPs [38, 48–51, 68–83]. For instance, it showed that the nature
of supports played a critical role in the catalytic activities of supported AuNPs as
GOx mimics [50]. Besides AuNPs-based GOx mimics, a few other metal nano-
materials have also been explored for mimicking GOx [52–54]. For example, Pd
nanoparticles on γ-Al2O3 support exhibited good selectivity toward glucose oxi-
dation [52]. It has also demonstrated that Au atom decorated Pd nanoparticles
exhibited higher catalytic activity toward glucose oxidation when compared with
AuNPs, PdNPs, and Pd/Au alloys [54]. It further demonstrated that Au-containing
bimetallic and trimetallic nanoparticles showed higher catalytic activities than
monometallic ones [53]. The enhanced activities have been ascribed to several
factors, such as electronic charge transfer effect among different metals, geometric
effect, and structural changes [53].
3.2.2 Metal Nanomaterials as Multiple Enzyme Mimics
As mentioned above, many metal nanomaterials have exhibited multiple enzyme

mimicking activities under different conditions [47, 65, 84–89]. For example,
PtNPs encapsulated within ferritin have exhibited both peroxidase and catalase
mimicking activities [84]. Due to the protection of the ferritin shell, the nanozymes
showed high stability. The peroxidase mimicking activity was confirmed by oxi-
dizing colorless substrates (i.e., TMB and DAB) with H2O2 into the corresponding
colored products in the presence of the nanozymes. The catalase mimicking activity
was established by producing oxygen bubbles from H2O2 decomposition and by
inhibiting the formation of hydroxyl radicals from H2O2 in the presence of the
nanozymes. Moreover, they exhibited peroxidase-like activity at slightly acidic
condition and physiological temperature. When the pH and reaction temperature
increased, the catalase-like activity dominated [84]. The SOD and oxidase mim-
icking activities have also been observed in several nanomaterials [47, 86–88].
To better understand the multiple enzyme mimicking activities of metal nano-
materials, Wu, Gao and co-workers have carried out detailed computational studies
[65, 89]. Au, Ag, Pt, and Pd nanomaterials exhibited pH-switchable peroxidase-like
and catalase-like activities (i.e., they were peroxidase and catalase mimics at acidic
and basic conditions, respectively) [89]. To unravel the origin of such
pH-switchable catalytic activities, the H2O2 adsorption and the H2O2 decomposi-
tion behaviors on metal surfaces were calculated. First, the calculation of H2O2
adsorption on Au(111) surface indicated that H2O did not prevent H2O2 adsorption
at neutral pH. At acidic pH, H would be pre-adsorbed, which in turn weakened the
H2O2 adsorption on Au slightly. At basic pH, H2O2 interacted with Au atoms in the
vicinities of the pre-adsorbed OH. As shown in Fig. 3.11a, it favored the base-like
decomposition pathway at neutral conditions, which would produce adsorbed H2O
(i.e., adsorbed H2O*) and adsorbed O (i.e., adsorbed O*). However, the adsorbed O
may not form O2 due to the high energy barrier of 1.42 eV. As shown in
Fig. 3.11B, at acidic conditions, it still preferred the base-like decomposition
pathway. Due to the presence of pre-adsorbed H, H2O2 would be decomposed into
an adsorbed H2O (i.e., adsorbed H2O*) and an adsorbed OH (i.e., OH*). The OH*
would be converted into H2O* and O*. The formed O* subsequently oxidized
organic substrates by abstracting H atom from them. Therefore, the Au(111) surface
would act as a peroxidase mimic at acidic conditions. On the other hand, at basic
conditions, it still preferred the acid-like decomposition pathway (Fig. 3.11c). In
this case, one H from H2O2 would be react with the pre-adsorbed OH to form H2O*
and HO*2. The newly formed HO*2 would transfer one H to another H2O2, resulting
the formation of H2O* and O*2. Therefore, the Au(111) surface would act as a
catalase mimic at basic conditions. The above-calculated results were consistent
with the experimental results [89].
Further calculation revealed that for both Au(110) and Au(211) surfaces, they
also favored base-like decomposition and acid-like decomposition pathway at
acidic and basic conditions, respectively. In other words, both of them acted as
peroxidase and catalase mimics at acidic and basic conditions, respectively. Since
the enzyme mimicking activities were not dependent on the specific surfaces, it
suggested that the catalytic activities were intrinsic properties. However, among the
three surfaces, Au(111) surface exhibited the least catalytic activities due to the
highest energy barriers [89].
To understand the different enzyme mimicking activities of different metal
nanomaterials, the H2O2 decomposition on Ag(111), Pt(111), and Pd(111) surfaces
was also calculated [89]. Several conclusions could be obtained from the calcula-
tion results. First, all of them followed the same reactions pathways as Au(111)
Fig. 3.11 pH-switchable enzyme-mimic activities of metals. Calculated reaction energy profiles
for H2O2 decomposition on the Au(111) surface in neutral (a), acidic (b) and basic (c) conditions
(unit: eV). d Relationships between adsorption energies (Eads) and activation energies (Eact) for
H2O2 decompositions on metal’s (111) surfaces at acidic (left) and basic (right) conditions. e TEM
images of the nanorods. Enzyme-mimic activities of Au@Pd nanorods (0.1 nM) at 0.1 m PBS
buffers with different pH values for peroxidase with f 20 mm H2O2 + 0.4 mm OPD and for
catalase with h 20 mm H2O2, respectively. Peroxidase-like activity at pH = 4.5 (g) and
catalase-like activity at pH = 7.4 (i) for the metals. Unless indicated, reaction temperature in (f–i))
is 30 °C. Reprinted from Ref. [89], Copyright 2015, with permission from Elsevier
surface did due to their structure similarities (i.e., they acted as peroxidase and
catalase mimics at acidic and basic conditions, respectively). Second, the activation
energies (Eact) of them for both mimics followed the order: Au(111) < Ag
(111) < Pt(111) < Pd(111). Third, the adsorption energies (Eads) and activation
energies (Eact) exhibited an approximate linear relationship (Fig. 3.11d). It also
suggested that the affinities of the metal surface toward H2O2 followed the order:
Au(111) < Ag(111) < Pt(111) < Pd(111). Therefore, Eads could be practically used
to estimate the relative catalytic activities of nanozymes [89].
The above calculations indicated that for both mimics, their catalytic activities
would follow the order: Au(111) < Ag(111) < Pt(111) < Pd(111). To test the
hypothesis, four nanorods (i.e., Au, Au@Ag, Au@Pt, and Au@Pd) were synthe-
sized and their activities were evaluated (Fig. 3.11e). First, the pH-switchable
activities were observed for Au@Pd nanorods (Fig. 3.11f and 3.11h). Second, for
different metal nanomaterials, both enzyme mimicking activities followed the order:
Au(111), Ag(111) < Pt(111), Pd(111) (Fig. 3.11g and 3.11i), which was consistent
with the calculations. Note that, the following orders were observed: Ag(111) < Au
(111) and Pd(111) < Pt(111). Such deviation between the experimental and cal-
culation results could be attributed to the following facts. For Ag(111), it could be
easily oxidized and therefore affected its activities. For Pt(111), it had larger surface
compared with Pd(111). Considering these facts, the calculations have provided
mechanistic insights of the metals’ enzyme mimicking activities and have provided
a way to predict the nanozymes’ activities [89].
Wu, Gao and co-workers also studied the origins of oxidase and SOD mimicking
activities of Au, Ag, Pt, Pd, and their alloys [65]. For the oxidase mimicking
activities, the adsorption of O2 and breakage of O–O bond (i.e., the dissociation of
O2) should be critical since it would transfer the magnetic moments from 3O2 to the
metal and thus allow originally spin forbidden 3O2 react with its substrate via O*.
For Au(111), Ag(111), Pt(111), and Pd(111), though O2 could be adsorbed onto all
of them and subsequently weakened the O–O bond of O2 in an energy favorable
manner, only Pt(111) and Pd(111) exhibited low Eact and negative Er (reaction
energy) values. The high Eact and positive Er values for Au(111) and Ag(111)
suggested that Pt(111) and Pd(111) would exhibit oxidase mimicking activities
while Au(111) and Ag(111) would not. Since the oxidase mimicking activities of
AuNPs have been reported [37], there should be some factors responsible for such
activities. To further reveal the origin of the AuNPs’ oxidase-like activities, the
calculations for Au(110) and Au(211) surfaces were also carried out. Though Au
(110) still did not show favorable oxidase mimicking activity, Au(211) did owing to
the low Eact and negative Er. It suggested that the oxidase mimicking activities of
the reported AuNPs might be originated from their high-energy facets, such as Au
(211) [65]. Low-spin state 1O2 could be converted from 3O2 by the surface plasmon
resonance of noble metals. Though the dissociation of 1O2 were kinetically easier
than that of 3O2, the Eact values were still too large for Au(111) and Ag(111)
surfaces. Therefore, the conversion of O2 from 3O2 to 1O2 would not substantially
affect the metal-mediated O2 dissociation and thus their oxidase-like activities [65].
By forming alloys, the oxidase-like activities could be significantly modulated
[65]. The calculation results indicated that the formation of AuAg alloys has sig-
nificantly reduced the Eact values compared with Au and Ag themselves. Moreover,
Er values for the alloys also became negatively. Therefore, the formation of AuAg
alloys would enhance the oxidase mimicking activities of both Au and Ag. In
contrast, the formation of alloys between Pd (or Pt) and Au led to the increase of
Eact values and the positive Er values [65]. Therefore, the oxidase mimicking
activities were disfavored for the AuPd or AuPt alloys. These results suggested a
possible approach to modulating the metals’ oxidase mimicking activities (in both
positive and negative manners).
Since O∙− − ∙
2 radical can easily react with H2O to produce OH and HO2, the SOD
mimicking activities of metal nanomaterials are mainly due to their capability for
rearrange HO∙2 into H2O2 and O2. The calculation results showed that the Eact
values for the rearrangement on both Au(111) and Pt(111) were very small, indi-
cating that both of the metals could act as high efficient SOD mimics [65].
3.2.3 Applications
Along with exploring the mechanisms of the metal-based nanozymes, they have
also been widely used for various applications [79, 80, 86, 90–96].
(a) Immunoassays
The metal nanomaterials-based oxidase, peroxidase, and even catalase mimics
have been employed for immunoassays [86, 91, 95, 97–101]. For instance, sandwich
assays for interleukin 2 (IL-2, a cytokine) have been developed with Au@Pt
nanorods [86]. As shown in Fig. 3.12, both the oxidase and peroxidase mimicking
activities of the Au@Pt nanorods could be employed for detection. In both cases,
colorless TMB would be oxidized into colored products when IL-2 was present [86].
When the nanozymes were conjugated with tumor cell targeting molecules, the
conjugates could be used for tumor cell immunoassay. For instance, when Au
nanoclusters decorated graphene oxide was labeled with folic acid, the conjugates
showed peroxidase-like activity and have been used for selectively quantifying
MCF-7 tumor cells [98]. Using Pt nanoparticles as catalase mimics, a volumetric
bar-chart chip has been developed for detection of biomarkers both in serum and on
cell surface [99]. An integrin specific peptide was used to modify AuNPs-based
peroxidase mimics. The obtained conjugates were then used as specific probes for
cancer detection [101].
(b) Glucose and other bioactive small molecules detection
By combing GOx with nanomaterials-based peroxidase mimics, glucose could
be detected [58, 64, 87, 102–104]. Due to high specificity of natural GOx, these
assays exhibited high selectivity toward glucose detection. Since AuNPs could act
as GOx mimics, they were combined with hemin (a peroxidase mimic) together for
colorimetric detection of glucose [81]. The selectivity of AuNPs-based GOx mimics
remained to be studied.
In principle, when a natural oxidase was combined with nanomaterials-based
peroxidase mimics, the corresponding oxidase substrate as the target of interest
could be determined (also see Tables A1 and A2). For example, a colorimetric
assay for cholesterol was developed by combining cholesterol oxidase with Pt
nanoparticles-based peroxidase mimics [64]. More interestingly, sarcosine, a
Fig. 3.12 Immunoassays for interleukin 2. a Au@Pt nanorods-based assays. b Detection of IL-2
using Au@Pt nanorods’ oxidase (i.e., without H2O2) or peroxidase (i.e., with H2O2) mimicking
activities. Reprinted from Ref. [86], Copyright 2011, with permission from Elsevier
potential biomarker for prostate cancer, in clinical samples has been successfully
determined by using sarcosine oxidase and Pd nanoparticles-based peroxidase
mimics [105].
(c) Detection of analytes by modulating nanozymes’ activities
The nanozymes’ activities could be modulated by changing their sizes, surface
coating, etc. Based on these phenomena, numerous strategies have been developed
for sensing analytes of interest [78, 106–113].
Fan, Li, and co-workers found that the catalytic activities of AuNPs-based GOx
mimics were controlled in a self-limited manner [107]. As discussed in Chap. 2,
ssDNA and dsDNA have different affinities toward AuNPs. By combing the
AuNPs’ GOx mimicking activities with DNA modulation, a general sensing plat-
form was developed (Fig. 3.13) [106]. The probe ssDNA would interact with
AuNPs nanozymes and thus inhibited their catalytic activities. The presence of
targets (such as a complementary ssDNA, a complementary miRNA, or an analyte
for an aptamer if the probe ssDNA was an aptamer), the probe ssDNA would form a
complex with its target and thus recover the activity of AuNPs. The nanozymes
activities were then monitored with suitable output signals. For example, when it
was coupled with HRP, either colorimetric or chemiluminescent signal would be
produced. The intrinsic plasmonoic signals of the AuNPs could also be used for
signaling [106]. Inspired by this work, several reports showed that other functional
nucleic acids (including aptamers) could be used to modulate the nanozymes’
activities [108, 109, 111, 114, 115]. For example, a colorimetric method for the
detection of kanamycin has been developed [108]. Kanamycin is an aminogly-
coside antibiotic widely used in veterinary medicine. Its aptamer would bind onto
AuNPs and inhibit their peroxidase-like activities. The specific interaction between
kanamycin and its aptamer would recover the inhibited activities of the nanozymes.
Via such a switchable strategy, kanamycin has been detected with good sensitivity
and selectivity. Using the same principle, Li et al. reported the colorimetric
detection of ricin with its aptamer and peroxidase-like AuNPs [115].
Besides DNA, other biomolecules could also be used to modulate the nano-
zymes activities and to develop biosensors [78, 113, 116]. Li and co-workers found
that melamine could significantly improve the peroxidase-like activity of AuNPs
[116]. By making use of such enhancement effect, they proposed a colorimetric
method for melamine detection (Fig. 3.14). The developed method was robust
Fig. 3.13 Illustration of the GOx-like catalytic activity of AuNPs regulated by DNA hybridiza-
tion, which can be either amplified by HRP cascaded color or chemiluminescence variations (path
a) or lead to nanoplasmonic changes owing to size enlargement (path b). Orange strand = target,
green strand = adsorption probe. Reprinted from Ref. [106], Copyright 2011, with permission
from John Wiley and Sons
Fig. 3.14 Detection of melamine based on its enhancement toward the peroxidase-like activity of
AuNPs. Reprinted from Ref. [116], Copyright 2014, with permission from Elsevier
Fig. 3.15 Detection of Hg2+ based on its inhibition toward the peroxidase-like activity of PtNPs.
Reprinted from Ref. [117], Copyright 2015, with permission from Elsevier
enough that it has been employed to detect spiked melamine in raw milk and milk
powder [116].
Metal ions have also been used to modulate the nanozymes’ activities [117,
118]. As shown in Fig. 3.15, the peroxidase-like activity of PtNPs could be
selectively inhibited by Hg2+ due to the specific aurophilic/metallophilic interac-
tions between Hg2+ and Pt0 [117]. Based on such specific inhibition, Hg2+ has been
selectively detected against several other metal ions (such as Na+, Mg2+, Ca2+,
Mn2+, Ni2+, Zn2+, Co2+, Cu2+, Pd2+, Cd2+, Fe3+, and Au3+).
(d) Other applications
Other potential applications of metal nanomaterials-based nanozymes have also
been explored [61, 119]. For instance, Qu, Ren, and co-workers have studied the
antibacterial activity of AuNPs encapsulated within mesoporous silica [119]. The
nanozymes exhibited both oxidase and peroxidase mimicking activities. Since ROS
(reactive oxygen species) species (such as O∙− ∙
2 and HO ) were involved in the
mimicking activities, it was reasonable to test the nanozymes’ antibacterial activity.
As expected, the nanozymes showed antibacterial properties against both
Gram-negative and Gram-positive bacteria [119].
References 49
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Chapter 4
Metal Oxide-Based Nanomaterials
for Nanozymes
Abstract Metal oxide-based nanomaterials have been extensively studied to mimic

various natural enzymes due to their unique properties. In this chapter, several metal
oxide-based nanozymes are discussed. First, the use of cerium oxide nanomaterials
for mimicking natural enzymes (such as superoxide dismutase, catalase, oxidase,
peroxidase, phosphatase, etc.) is discussed. Second, the use of iron oxide nano-
materials for peroxidase mimics and other mimics is covered. Third, the enzyme
mimicking activities of other metal oxides (such as vanadium oxide, cobalt oxide,
copper oxide, etc.) are discussed. The catalytic mechanisms are also discussed if
they have been elucidated. Selected examples for broad applications are discussed,
which cover from glucose detection, DNA detection, immunoassay, and
immunostaining, to neuroprotection, cardioprotection, cancer therapy, and tissue
engineering.
Keywords Nanozymes
Artificial enzymes
Enzyme mimics Metal

oxide-based nanomaterials Cerium oxide Iron oxide Reactive oxygen species

Oxidase mimics Glucose detection Brain chemistry
Metal oxide-based nanomaterials (such as cerium oxide and iron oxide) have been
extensively studied to mimic various natural enzymes. These nanozymes have
found broad applications in many areas, from bioanalysis to therapeutics. In this
chapter, we will discuss the nanozymes based on these metal oxide nanomaterials.
4.1 Cerium Oxide
Cerium oxide nanomaterials (also called nanoceria) have been widely used as
highly efficient catalysts due to their unique properties (i.e., the presence of mixed
valence states of Ce3+ and Ce4+ and highly mobile lattice oxygen) [1]. They have
also been extensively explored for mimicking natural enzymes [2–8]. In 2005,
Tarnuzzer, Seal and co-workers reported that vacancy engineered nanoceria could

58 4 Metal Oxide-Based Nanomaterials for Nanozymes
protect normal but not tumor cells from radiation-induced damage [9]. Since then, a
large number of studies have showed that nanoceria could mimic SOD, catalase,
oxidase, peroxidase, phosphatase, etc. [10–35].
4.1.1 Cerium Oxide as SOD Mimics
(a) Catalytic activities and mechanisms

In their seminal study, Tarnuzzer, Seal and co-workers attributed the protective
role of the nanoceria to its free radicals elimination capability. It was suggested that
the radicals could be eliminated by nanoceria via a Ce3 þ ! Ce4 þ ! Ce3 þ
regeneration mechanism [9]. Self, Seal, and co-workers as well as other groups
have then established the SOD mimicking activities of nanoceria [36–38]. Based on
the competitive cytochrome C assay, H2O2 formation assay, and EPR measure-
ments, Self et al. [36, 37] proposed that the nanoceria would exert the SOD-like
activities via the mechanisms shown in Eqs. 4.1–4.2. A possible molecular
mechanism was proposed (Fig. 4.1) [39]. However, more detailed studies are still
needed to confirm (or even modify) the proposed mechanism.
Fig. 4.1 Proposed molecular mechanisms for the SOD mimicking activity of nanoceria.
Reprinted from Ref. [39], Copyright 2011, with permission from Royal Society of Chemistry
4.1 Cerium Oxide 59
O
2 þ Ce
4þ
! O2 þ Ce3 þ ð4:1Þ
O
2 þ Ce
3þ
þ 2H þ ! H2 O2 þ Ce4 þ ð4:2Þ
The SOD-like catalytic activity of nanoceria was dependent on the redox state of
surface cerium ions (i.e., the SOD-like activity was positively correlated with the
ratio of Ce3+/Ce4+) [36, 37]. It was also found that the SOD mimicking activity of
nanoceria was size dependent. Nanoceria larger than 5 nm did not show prominent
SOD activity [36]. An interesting strategy has been proposed to endow the
nanoceria larger than 5 nm with SOD mimicking activities [40]. When electrons
were transferred either from a native SOD or other donors (e.g., an inorganic metal
complex) to the nanoceria with large size, its superoxide-scavenging capability was
restored. The transferred electrons would reduce Ce4+ to Ce3+ and thus improve the
SOD-like activity [40]. The SOD mimicking activities of nanoceria could be
modulated by other strategies [41–43]. For example, by doping with nanoceria with
redox inactive Sm and Ti atoms, its catalytic activity could be reduced [41, 43].
(b) Applications
Nanoceria-based SOD mimics have been used for various applications, ranging
from anti-inflammation and neuroprotection to tissue engineering and cancer
therapy [2, 44–46].
Neuroprotection. Numerous studies have demonstrated that nanoceria had
neuroprotection functions [47–50]. In their pioneering study, McGinnis et al.
showed that nanoceria could protect rat retina photoreceptor cells from light-induced
degeneration (Fig. 4.2). The protection activity was probably due to the elimination
of ROS (reactive oxygen species) by nanoceria. Encouragingly, the nanozymes
showed therapeutic effects when they were administrated either before or even after
light exposure [47]. In a later report, they showed that nanoceria exhibited long-term
therapeutic effects of protecting photoreceptor cells from degeneration [48]. Using
the very low density lipoprotein receptor knockout (vldlr–/–) mouse as a model,
McGinnis et al. [49] demonstrated that nanoceria was effective against age-related
macular degeneration diseases.
Other groups have also demonstrated the neuroprotection roles of nanoceria in
several disease models, such as adult rat spinal cord injury, brain ischemia,
Alzheimer’s disease, autoimmune encephalomyelitis, etc. [51–55]. For example,
using mouse hippocampal brain slice as an in vitro model of brain ischemia, it has
showed that the treatment of nanoceria could significantly reduce the ischemic cell
death (Fig. 4.3) [52]. Detailed studies revealed that the concentrations of super-
oxide (O∙−
2 ) and nitric oxide were reduced by around 15 %. Moreover, the level of
peroxynitrite-induced 3-nitrotyrosine was remarkably reduced by 70 %. Therefore,
it was proposed that the nanozymes may protect the brain cells from ischemic injury
by scavenging ROS and NOS (such as peroxynitrite, O∙− 2 , and nitric oxide) [52].
The nanozyme even protected cell from ischemic injury in living mice brains [53].
Fig. 4.2 Intravitreal injection of nanoceria protected rat retina photoreceptor cells from
light-induced degeneration. Reprinted from Ref. [47], Copyright 2006, with permission from
Nature Publishing Group
To further improve the therapeutic efficacy, one may conjugate specific recognition
moieties onto the nanozymes for targeted therapy [55].
Cardioprotection. A few studies have showed that nanoceria possessed car-
dioprotective activities [56–58]. Traversa, Nardo, and co-workers showed that
nanoceria protected cardiac progenitor cells, a valuable cell source for cardiac
regenerative medicine, from oxidative stress [56]. For an in vivo mouse model with
ischemic cardiomyopathy, the treatment with nanoceria effectively inhibited the
progressive left ventricular dysfunction and dilatation. The therapeutic benefits of
the nanozymes were due to the inhibition of myocardial oxidative stress, ER stress
and inflammatory processes, which were evidenced by the decrease of
pro-inflammatory cytokines (such as tumor necrosis factor-α and interleukin-1β)
and downregulation of endoplasmic reticulum stress-associated genes [59]. Heart
hypertrophy following pulmonary arterial hypertension could also be attenuated by
nanoceria treatment [58].
Cancer therapy. Researchers have used nanoceria to treat cancers [60–63].
Cellular studies demonstrated the feasibility of using nanoceria for cancer therapy.
Due to the Warburg effect, the nanozyme would benefit stromal cells by eliminating
the elevated ROS. At the same time, the nanozyme would inhibit tumor cells’
metastatic spread by inhibiting the myofibroblasts formation, etc. [60]. The in vivo
anticancer activities of the nanoceria were evaluated using mice with xenografted
melanoma. The treatment with nanoceria led to significantly smaller tumor volume
4.1 Cerium Oxide 61
Fig. 4.3 Neuroprotective effect of nanoceria treatment after brain ischemia. Nanoceria signifi-
cantly decreased the area of ischemia-induced cell death in hippocampal slices. a Pseudocolor
images of brain slices loaded with Sytox blue. b Quantification of the area of Sytox fluorescence in
mouse hippocampal slices after 30 min of ischemia and treatment with varying doses of nanoceria.
Reprinted from Ref. [52], Copyright 2011, with permission from Elsevier
and weight, thus showing the translational promise of the nanoceria [61]. It was also
reported that nanoceria could inhibit ovarian tumor growth via an anti-angiogenic
mechanism [62].
Tissue engineering. Nanoceria has been used to promote (stem) cell prolifera-
tion and tissue engineering [64–68]. Traversa et al. prepared hybrid materials with
enhanced mechanical properties by fabricating nanoceria together with PLGA
scaffolds. They went on to demonstrate that the hybrids could promote the
murine-derived cardiac and mesenchymal stem cells growth. Initially, the promo-
tion effects of nanoceria were attributed to its antioxidation properties [64].
However, later they revealed that the cell proliferation was more favored on Ce4+
dominant regions rather than on Ce3+ dominant regions [65]. It suggested that Ce4+
dominant regions would promote the cell proliferation due to its hydrophilicity. On
the other hand, Ce3+ dominant regions inhibited the cell proliferation by limiting
Fig. 4.4 Specific cell growth process on Ce4+and Ce3+ regions. Reprinted from Ref. [65],
Copyright 2014, with permission from Elsevier
cell spreading and weakening the cell–material interactions (Fig. 4.4) [65]. This
result was in agreement with other reports, which suggested that surface Ce3+ might
be biotoxic [69].
Mattson, Seal, and co-workers demonstrated that nanoceria could accelerate
cutaneous wound healing in mice by promoting the growth of keratinocytes,
fibroblasts, and vascular endothelial cells. The therapeutic efficacy was owing to the
enhanced proliferation and migration of these cells [66]. As shown in Fig. 4.5,
when nanoceria (prepared in water) was used as additives, they could promote bone
regeneration by enhancing collagen production from human mesenchymal stem
cells. In comparison with the scaffolds themselves, osteoblast-like products were
observed on the nanoceria-doped scaffolds even in the absence of osteogenic
supplements. The promotion mechanism still remained unclear [67].
Anti-inflammation and antioxidation. Owing to the mixed valence and oxygen
vacancy, nanoceria-based nanozymes exhibited anti-inflammation and antioxida-
tion effects. Using J774A.1 murine macrophage cells, Reilly et al. demonstrated
that nanoceria possessed anti-inflammatory activities. The nanoceria exhibited good
biocompatibility after being internalized by the cells. Moreover, the nanoceria could
both scavenge ROS and inhibit NO production, which in turn would inhibit
inflammation [70]. In a mouse model with CCl4-induced liver injury, it was showed
that the treatment with nanoceria mitigated systemic inflammation, reduced hepatic
steatosis, and improved portal pressure. The anti-inflammatory effects of the
nanoceria were also evidenced by the remarkable downregulation in mRNA
expression of inflammatory cytokines, vasoconstrictor, oxidative stress messengers,
etc. This study indicated that nanoceria might be used as a therapeutic drug for liver
disease [71]. Nanoceria has also been widely used as antioxidants [41, 56, 72, 73].
Its antioxidizing activity was even better than that of commercial antioxidants such
as Trolox [73].
Other applications. Nanoceria-based SOD mimics have been explored for other
interesting applications [74, 75]. For instance, nanoceria showed antibacterial
activities against Escherichia coli (a typical Gram-negative bacterium) and Bacillus
subtilis (a typical Gram-positive bacterium) [74]. More interestingly, it showed that
the nanoceria was ineffective against Shewanella oneidensis, which was not only a
Gram-negative bacterium but also a metal-reducing bacterium. These studies
indicated that the antibacterial activities of nanoceria were dependent not only on
4.1 Cerium Oxide 63
Fig. 4.5 SEM images of human mesenchymal stem cells cultured for 10 days on the bioactive
glass scaffolds doped with nanoceria synthesized in water (a, b), in dextran (c, d) and without
nanoceria (e, f). Cell culture was performed in the absence (a, c, e) and in the presence (b, d, f) of
osteogenic supplements. Cells attached and spread on the scaffolds’ surface. Note the
osteoblast-like products or minerals on the cells cultured in the absence (a, c, e) of osteogenic
supplements (scale bar = 10 μm). Reprinted from Ref. [67], Copyright 2010, with permission
from Royal Society of Chemistry
the nanozyme’s intrinsic properties (such as size, surface coating, etc.) but also on
the bacterial species.
Recently, nanoceria was integrated with a bioresorbable electronic stent [76].
During percutaneous coronary interventions, it might produce ROS and cause
in-stent thrombosis due to the inflammation. To address these issues, nanoceria was
used to not only scavenge ROS but also mitigate inflammation.
4.1.2 Cerium Oxide as Catalase Mimics
Besides the SOD mimicking activities, the catalase-like activities of nanoceria have
also been studied [77–80]. It was found that the oxidase mimicking activity of
nanoceria was negatively correlated with the ratio of Ce3+/Ce4+, which was different
from its SOD mimicking activity [78]. Detailed experimental and computational
studies revealed that nanoceria could dismutate H2O2 into H2O and O2 via the
mechanism shown in Eqs. 4.3–4.7 [79]. Such a mechanism was also consistent with
a previously proposed one (Fig. 4.6) [39].
2Ce3 þ þ H2 O2 þ 2H þ ! 2H2 O þ 2Ce4 þ ð4:3Þ
H2 O2 þ Ce4 þ ! Ce3 þ þ 2H þ þ HOO ð4:4Þ
Ce4 þ þ HOO ! Ce3 þ þ H þ þ O2 ð4:5Þ
H2 O2 þ 2Ce4 þ ! 2Ce3 þ þ 2H þ þ O2 ð4:6Þ
2H2 O2 ! 2H2 O þ O2 ð4:7Þ
Fig. 4.6 Proposed molecular mechanisms for the catalase mimicking activity of nanoceria.
4.1 Cerium Oxide 65
Traditional bioactive glasses with the composition of Na2O, CaO, SiO2, and
P2O5 have been used for bone defect reparation. However, they cannot effectively
prevent oxidative stress and reduce inflammation after implantation due to the inert
chemistry. By introducing redox active nanomaterials, especially the ones with
antioxidation and anti-inflammation activities, into the bioactive glasses would
address this issue. To this end, nanoceria has been used to dope traditional bioactive
glasses. The catalase mimicking activity of nanoceria-doped glasses was confirmed
by in vitro experiments and computational studies [79].
Liu et al. [77] developed an interesting strategy to fabricate biosensors for H2O2
and glucose. As shown in Fig. 4.7, the adsorption of dye-labeled DNA onto
nanoceria quenched its fluorescence. The presence of H2O2 would displace
Fig. 4.7 a Sensing H2O2 by displacing adsorbed fluorescent DNA from nanoceria. The color of
nanoceria is changed in the same process. b A proposed mechanism of H2O2-induced DNA release
by capping the nanoceria surface. For the three time scales marked in the scheme, DNA release is
related to the one on the order of 1 min. Adapted from Ref. [77], Copyright 2015, with permission
from American Chemical Society
adsorbed DNA from nanoceria and thus recover its fluorescence. The possible
sensing mechanism was showed in Fig. 4.7b. The phosphate group of the DNA
interacted with the Ce3+ on the nanoceria surface. H2O2, however, could compete
against the bound DNA by oxidizing the Ce3+ into Ce4+ within one minute. The
bound H2O2 would then be converted into H2O and O2 via a catalase-like mech-
anism. Moreover, when glucose oxidase (GOx) was coupled with the H2O2-
mediated DNA displacement from nanoceria, sensitive and selective glucose
detection was achieved [77]. Using the same strategy, cellular H2O2 was detected
with cerium oxide nanowires [80].
4.1.3 Cerium Oxide as Peroxidase Mimics
Nanoceria has been successfully used to mimic peroxidase [5, 81, 82]. The per-
oxidase mimicking activities were confirmed by the fact that nanoceria could cat-
alyze peroxidase’s substrates with H2O2. The detection of H2O2 and glucose has
been achieved by making use of the nanoceria’s peroxidase-like activities [5].
Moreover, a sandwich immunoassay has been developed for detection of a breast
cancer biomarker (i.e., CA15-3) [81]. The peroxidase-like nanoceria was attached
to the detection antibody electrostatically, eliminating the time-consuming covalent
bioconjugation procedure. Compared with the natural HRP-based sandwich assay,
the nanozyme-based assay not only exhibited higher stability and lower cost but
also had higher sensitivity (i.e., it had a detection limit one order of magnitude
lower than that of HRP-based assay) [81].
4.1.4 Cerium Oxide as Oxidase Mimics
Perez et al. [83] reported the oxidase mimicking activity of polymer-coated

nanoceria. Since no H2O2 was involved, the observed catalytic activity toward
TMB oxidation at acidic conditions was attributed to the oxidase-like rather
peroxidase-like properties of the nanoceria. The size-dependent study showed that
smaller nanoceria exhibited higher oxidase mimicking activities. Moreover, the
catalytic activities were also dependent on the surface coatings. The thinner and
more permeable poly(acrylic acid)-coated nanoceria exhibited higher activity in
comparison with the nanoceria coated with thicker dextran. Interestingly, when
nanoceria was conjugated with folate, the obtained nanozyme probes were used for
specific cancer cell detection (Fig. 4.8) [83]. The developed immunoassay was
advantageous over traditional ELISA in the eliminating the use of unstable H2O2
and easily denatured HRP. The oxidase mimicking activities of nanoceria could be
regulated by binding with ssDNA [84]. A colorimetric method for DNA detection
has been reported by using the oxidase-like nanoceria. The detection was based on
the target DNA induced shielding of the catalytic activity of nanozyme [85].
4.1 Cerium Oxide 67
Fig. 4.8 Comparison of traditional ELISA (a) and nanoceria-based ELISA (b). In traditional
ELISA, an HRP antibody is utilized as secondary antibody that, upon hydrogen peroxide
treatment, facilitates the oxidation of TMB, resulting in color development. In nanoceria-based
ELISA, the oxidase-like activity of nanoceria facilitates the direct oxidation of TMB without the
need of HRP or hydrogen peroxide. Reprinted from Ref. [83], Copyright 2009, with permission
from John Wiley and Sons
4.1.5 Cerium Oxide as Other Mimics
Nanoceria has also been explored for mimicking other enzymes [18, 86, 87]. For
example, nanoceria was able to eliminate nitric oxide radical. Unexpectedly, the
nitric oxide radical scavenging activity of nanoceria was negatively correlated with
the Ce3+/Ce4+ ratio, which was different from its SOD-like activity [86].
Kuchma et al. found that the nanoceria exhibited phosphatase-like activities.
Nanoceria promoted the hydrolysis of phosphate ester bonds in p-nitrophenyl-
phosphate (pNPP), o-phospho-L-tyrosine, ATP, but not in DNA. They showed that
the phosphatase mimicking activity was dependent on the Ce3+ sites and oxygen
vacancies. Facilitated with first principles calculation, it was revealed that the
catalysis proceeded via a SN2 mechanism (Fig. 4.9). Moreover, the activation
energy for the hydrolysis was significantly reduced when it was mediated by a Ce3
+
-Ce3+ complex instead of a Ce4+-Ce4+ complex (from 22.9 to 13.6 kcal/mol). The
lowered activation energy was attributed to the less polarized phosphorus-oxygen
bonds in the phosphate-Ce3+-Ce3+ complex, which was purely electronic. It also
suggested that the disfavored DNA hydrolysis with the nanoceria might be due to
the steric hindrance, which could shield the interaction of phosphate bond with the
Ce3+ sites [18].
Fig. 4.9 The snapshots of the reaction pathway for pNPP hydrolysis catalyzed by nanoceria.
a The reaction complex includes the neutral water molecule in the secondary coordination sphere.
b The hydrogen bonding is switched to the bridging hydroxide anion. c The proton is transferred
along this newly formed hydrogen bond. d The formed uncoordinated hydroxide then attacks
pNPP and e forms the transition state. f The p-nitrophenyl anion then leaves, g deprotonates one of
the water molecules in the first coordination sphere, and h forms the hydrogen-bonded product
complex. Reprinted from Ref. [18], Copyright 2010, with permission from Elsevier
4.2 Iron Oxide
Owing to their superior magnetic properties, iron oxide nanomaterials have been
widely used in bioanalytical and biomedical fields for separation and capture of
analytes, etc. Before Yan and co-workers’ surprising discovery, it was believed that
these nanomaterials were chemically and biologically inert. Therefore, they were
usually conjugated with various functional groups (such as metal catalysts,
enzymes, or antibodies) for practical applications. In 2007, Yan et al. discovered the
unexpected peroxidase mimicking activity of Fe3O4 magnetic nanoparticles
(MNPs) [88]. Inspired by this seminal work, quite a lot of nanomaterials (including
the iron oxide nanomaterials) have been explored to study their enzyme mimicking
activities [6, 7, 89–115].
Most of iron oxide nanomaterials have showed intrinsic peroxidase mimicking
activities though they could also mimic other natural enzymes. In this section, the
enzyme mimetic properties of iron oxide nanomaterials and their applications are
covered.
4.2.1 Iron Oxide as Peroxidase Mimics
(a) Catalytic activities and mechanisms

Natural peroxidases catalyze the oxidation of its substrates with peroxide (H2O2
in most of case). They play important roles in biological systems. For example,
4.2 Iron Oxide 69
Fig. 4.10 Fe3O4 MNPs performed as peroxidase mimics. a TEM images of employed Fe3O4
MNPs with different sizes. b The colorless peroxidase substrates TMB, DA and OPD were oxidized
into their corresponding colored products in the presence of H2O2 via the catalysis of Fe3O4 MNPs.
Reprinted from Ref. [88], Copyright 2007, with permission from Nature Publishing Group
glutathione (GSH) peroxidase can detoxify ROS while myeloperoxidase can defend
against pathogens. Moreover, HRP is widely used in bioanalytical and biomedical
research for amplifying detection signals, where it is conjugated to an antibody or
other biorecognition molecules. In their pioneering study, Yan et al. showed that
Fe3O4 MNPs could catalyze the oxidation of several peroxidase substrates with
H2O2, suggesting that they possessed intrinsic peroxidase mimicking activities
(Fig. 4.10) [88]. Compared with natural HRP, the nanozymes were unique in
several aspects. First, they were more stable and could work in wider pH and
temperature conditions. Second, they could be produced in large scale with low
cost. Third, they could be conjugated with biorecognition (or other functional)
elements due to the large surface area and rich surface chemistry. Fourth, they were
multifunctional (i.e., they had both magnetic and catalytic properties) [88].
Fe3O4 MNPs with different sizes (30, 50 and 300 nm) were studied, showing
that smaller nanoparticles had higher catalytic activity. Enzyme kinetics study
revealed that the Fe3O4 MNPs mimicked reactions also followed a ping-pong
mechanism (i.e., they exhibited parallel double-reciprocal plots of substrate con-
centration versus reaction rate) [88]. Interestingly, the nanozyme and HRP had Km
values of 0.098 mM versus 0.434 mM for TMB, respectively, which indicated that
the nanozyme showed even higher affinity to TMB in comparison with HRP. On
the other hand, the nanozyme exhibited lower affinity to H2O2 compared with HRP
[88]. The exact molecular mechanisms for the Fe3O4 MNPs mimicking peroxidase
activities are still unclear. A few studies have suggested that Fenton and/or Haber–
Weiss reaction mechanisms could be involved [116–118].
(b) Applications
Iron oxide nanomaterial-based nanozymes have been applied for many inter-
esting applications, ranging from detection of H2O2 and glucose to immunoassay
and immunostaining.
H2O2 detection. Detection of H2O2 is very important because it plays critical
roles in biology, medicine, environmental protection, food industry, etc. Wei and
Wang have reported the first example of H2O2 detection using Fe3O4 MNPs as the
peroxidase mimic and ABTS as the colorimetric substrate (Fig. 4.11a) [119, 120].
The presence of H2O2 could be easily visualized by the colored product of ABTSox
(i.e., oxidized ABTS∙+). Moreover, the amount of H2O2 could be quantified by
measuring the corresponding absorption spectra [119]. Since then, considerable
studies have been devoted to the H2O2 detection using peroxidase-like nanozymes
(see Table A.1) [6, 7].
Fig. 4.11 a Nanozyme as peroxidase mimic for colorimetric sensing of H2O2, and glucose when
combined with glucose oxidase. b The sensing format in (a) could be extended to other targets
(substrate 1 here) when combined with a suitable oxidase. c Target of interest as substrate 0 could
be determined if it could be converted into an oxidase substrate. Numerous transduction signals
can be adopted for sensing (such as colorimetric, fluorometric, chemiluminescent, and SERS
signals when the corresponding substrates are used; and electrochemical signals when a nanozyme
is immobilized on an electrode). Reprinted from Ref. [7], Copyright 2016, with permission from
Royal Society of Chemistry
4.2 Iron Oxide 71
Glucose (and other oxidase substrate) detection. Wei and Wang [119] went on
to demonstrate that glucose detection could be achieved by coupling GOx with a
peroxidase-like nanozyme. As shown in Fig. 4.11a, GOx first converted glucose to
H2O2, which subsequently oxidized a substrate into the corresponding product
(such as colored ABTS∙+ here). The established method showed excellent sensi-
tivity and selectivity toward glucose detection (Fig. 4.12) [119]. Followed by this
work, others have used other nanomaterials-based peroxidase mimics to detect
glucose even in complicated samples (such as in serum, urine, drinks, etc.) (see
Table A.2 for more examples) [6, 7].
As indicated in Fig. 4.11b, when other oxidases were used, the corresponding
oxidase substrates could be detected [6, 7]. Numerous biological important small
molecules, such as choline, uric acid, D-alanine, lactate, and xanthine, have been
detected with the proposed sensing method (see Table A.2 for more examples) [6, 7].
As indicated in Fig. 4.11c, the analytes as substrate 0 could also be detected by
converting them to oxidase substrates [6, 7]. Moreover, the proposed sensing
strategy could also be used to screen enzyme inhibitors. For example, Yan et al.
[121] reported a rapid and sensitive method for detecting organophosphorus pes-
ticides and nerve agents (such as Sarin), which were high potent acetyl-
cholinesterase inhibitors.
DNA detection. Two possible approaches are available for DNA detection by
using the peroxidase mimicking activities of iron oxide nanomaterials. First, it has
been established that ssDNA and dsDNA exhibited different affinities toward the
Fig. 4.12 Colorimetric detection of glucose by combining GOx with Fe3O4 MNPs as a
peroxidase mimic. Reprinted from Ref. [119], Copyright 2008, with permission from American
Chemical Society
nanozymes. A probe ssDNA would adsorb onto a nanozyme and thus shield its
activity. The presence of a target ssDNA would form a duplex with the probe
ssDNA, which would subsequently recover the nanozyme’s activity. For instance,
Park et al. reported the detection of Chlamydia trachomatis pathogen DNA from a
human urine sample via such a sensing strategy [122]. Second, by using a nano-
zyme as an alternative to conventional dyes (or enzymes) to label a probe ssDNA,
the target DNA could be detected via by hybridizing the probe ssDNA and target
ssDNA. Many sensing platforms could be adopted for this purpose. For example, in
a sandwich assay, a capture ssDNA would first interact with a target ssDNA. Then a
nanozyme labeled probe ssDNA would further interact with the captured target
ssDNA for signaling [123].
Liu et al. [124] found that in certain cases, DNA could even accelerate the
peroxidase mimicking activities of Fe3O4 MNPs. It was speculated that the
enhancement might be due to the electrostatic interaction between negatively
charged DNA and positively charged TMB substrate.
Immunoassay. In Yan’s initial report, they developed two immunoassay formats
[88]. Using an antigen-down immunoassay format, the detection of hepatitis B virus
surface antigen (preS1) was achieved. In a second demonstration, a capture-
detection sandwich immunoassay format was used for the detection of the
myocardial infarction biomarker troponin I (TnI) [88]. Yan and co-workers recently
developed a nanozyme-strip for Ebola detection by integrating lateral flow tech-
nique with the Fe3O4 MNPs nanozyme (Fig. 4.13) [125]. AuNPs are commonly
used to fabricate lateral flow strips, but their sensitivity should be further improved
to meet the requirements for infectious diseases (such as Ebola) diagnosis and
monitoring. To tackle this challenge, Yan et al. used Fe3O4 MNPs nanozyme to
replace AuNPs for fabricating the nanozyme-strips. Compared with the
AuNPs-based strip, the nanozyme exhibited 100-fold better sensitivity toward the
detection of Ebola virus glycoprotein (EBOV-GP) (Fig. 4.13c–e). The enhanced
sensitivity could be attributed to the high catalytic activity of the nanozymes.
Moreover, the nanozyme-strip showed comparable analytical performance with
ELISA. Due to high sensitivity, low fabrication cost, and ease of use, the
nanozyme-strip would find applications in fighting against emergent virus diseases,
especially in resource limited areas [125].
Other important targets, such as carcinoembryonic antigen (CEA), has been
detected by using Fe3O4 MNPs-based peroxidase mimics (see Table A.3 for more
examples) [126]. Cancer cells have also been selectively detected when the nano-
zymes were labeled with specific biorecognition elements (such as folic acid) [127].
Immunostaining. Yan et al. [99] also developed nanozymes for specific tumor
tissue imaging (Fig. 4.14). The probe was prepared by encapsulating catalytic iron
oxide nanoparticles within recombinant human heavy-chain ferritin shells to get
magnetoferritin nanoparticles. The magnetoferritin nanoparticles exhibited peroxi-
dase mimicking activity. When they were used for staining tumor tissues, they
4.2 Iron Oxide 73
Fig. 4.13 a Standard AuNPs-based strip, b nanozyme-strip employing Fe3O4 MNPs in place of
AuNPs, c nanozyme-strip, d standard colloidal gold strip and e ELISA method for EBOV-GP
detection. The asterisk (*) indicates the limit of visual detection of the test line in strips.#
OD450 nm > cut-off value. Reprinted from Ref. [125], Copyright 2015, with permission from
Elsevier
would differentiate tumor tissues from the benign ones due to the specific inter-
actions between ferritin and the overexpressed transferrin receptor 1 onto tumor
tissues. Very encouragingly, for the 474 patient specimens stained, the magneto-
ferritin nanoparticles distinguished cancer samples from normal ones with a
remarkable success (i.e., with a sensitivity of 98 % and a specificity of 95 %). Gu
and co-workers also showed that iron oxide nanoparticles-based nanozymes could
be used for immunohistochemical studies [94].
Aptasensors. Several studies have demonstrated that aptasensors could be
developed by combining the specific recognition capability of aptamers and the
Fig. 4.14 Magnetoferritin nanoparticles as peroxidase mimic for tumor tissue staining and
imaging. a Preparation of magnetoferritin nanoparticles. b Magnetoferritin nanoparticle staining of
tumor tissues. Reprinted from Ref. [99], Copyright 2012, with permission from Nature Publishing
Group
high catalytic activities of iron oxide-based nanozymes [128–130]. For instance,

Yang et al. [128] reported an aptasensor for thrombin. It is well known that
thrombin has two specific binding aptamers, one is a 15-mer and the other is a
29-mer. The two aptamers bind to the different sites onto a thrombin and thus would
form a sandwich structure. Based on such specific interaction, the 29-mer aptamer
was used to capture thrombin while the 15-mer aptamer was used to label iron
oxide nanoparticles. The presence of thrombin would form a sandwich structure,
and the 15-mer aptamer labeled iron oxide nanoparticles would catalyze the oxi-
dation of TMB for signaling [128]. Zhu, Wang, and co-workers [129] developed a
peroxidase mimic based on hybrid nanostructures (Fig. 4.15a). The nanozyme had
a Fe3O4 core, coated with Ag–Pd nanocages. Electrochemical measurements
demonstrated that the hybrid nanostructures had higher catalytic activity than Fe3O4
alone. They then developed an aptasensor for CTCs (circulating tumor cells)
4.2 Iron Oxide 75
Fig. 4.15 a Schematic illustration of the fabrication of Fe3O4@Ag–Pd nanozymes. b Schematic

illustration of CTCs detection. c DPV responses to different concentrations of MCF-7 cells. d DPV
responses to various types of cells. Reprinted from Ref. [129], Copyright 2014, with permission
from American Chemical Society
detection. SYL3C aptamer, which specifically interact with overexpressed epithelial

cell adhesion molecule (EpCAM) on CTCs, was used to capture the CTCs and to
label the hybrid nanostructures (Fig. 4.15b). As shown in Fig. 4.15c, d, the
developed aptasensor showed good sensitivity and selectivity toward CTCs
detection [129].
4.2.2 Iron Oxide as Other Enzyme Mimics
Iron oxide nanoparticles have been used to mimic catalase as well as other enzymes
[108, 131–135]. For instance, Gu and co-workers [131] found that iron oxide
nanoparticles exhibited dual enzyme-like activities. For both Fe3O4 and γ-Fe2O3
nanoparticles, they showed peroxidase-like and catalase-like activities at acidic and
neutral conditions, respectively. It was found that both the peroxidase and catalase
mimicking activities of Fe3O4 nanoparticles were higher than that of γ-Fe2O3
nanoparticles. The cellular studies revealed that the iron oxide nanoparticles
exhibited concentration-dependent cytotoxicity to human glioma U251 cells. The
cytotoxicity was attributed to the nanoparticles’ peroxidase-like activities within
acidic lysosomes, which would trap the nanoparticles to catalytically produce
hydroxyl radicals (Fig. 4.16). It also suggested that γ-Fe2O3 would be safer for
biomedical applications [131].
In a subsequent study, Gu and co-workers [133] went on to use Fe2O3
nanoparticles to protect hearts from ischemic damage both in vitro and in vivo
(Fig. 4.17a). It revealed that the protective activity was mainly from the nanopar-
ticles themselves rather than the surface coatings. Ischemic injury is mediated via
complicated mechanisms. Therefore, comprehensive studies were carried out,
which suggested that the Fe2O3 nanoparticles would exert their protection effects
via several mechanisms. First, they could inhibit the cellular ROS-induced mem-
brane lipid peroxidation. Second, they could attenuate Ca2+ influx, which in turn
Fig. 4.16 Schematic illustration of peroxidase-like activity-induced cytotoxicity by iron oxide

nanoparticles. The nanoparticles are trapped in acidic lysosomes when internalized into cells, so
they catalyze H2O2 to produce hydroxyl radicals through peroxidase-like activity; however, in
neutral cytosol, the nanoparticles would decompose H2O2 through catalase-like activity. Reprinted
from Ref. [131], Copyright 2012, with permission from American Chemical Society
4.2 Iron Oxide 77
Fig. 4.17 a Cardioprotective activity of Fe2O3 nanoparticles. b Effects of dietary Fe3O4

nanoparticles in a Drosophila Alzheimer’s Disease model. a Reprinted from Ref. [133], Copyright
2015, with permission from Nature Publishing Group. b Reprinted from Ref. [132], Copyright
2016, with permission from John Wiley and Sons
inhibited ROS. Third, they could increase NO production by promoting NO pro-

ducing proteins’ activities and enhance NO protective effects by increasing the level
of S-nitrosothiols. More convincing investigations are needed to completely
understand the protective roles of the Fe2O3 nanoparticles [108]. Nevertheless,
since the nanoparticles did not show obvious toxicity toward normal cardiomy-
ocytes and they were more potent than Verapamil (a synthetic drug) and Salvia
miltiorrhiza extract (a natural antioxidant), they were expected to be further
explored as potential nanomedicine for cardiovascular diseases treatment [133]. The
protective effects of iron oxide nanoparticles against ischemic brain injury were also
reported [134].
Dietary Fe3O4 nanoparticles have been used to treat Drosophila with Alzheimer’s
Disease (Fig. 4.17b) [132]. It was demonstrated that the Fe3O4 nanoparticles could
mimic catalase in vivo. Therefore, they would improve neurodegeneration in a
Drosophila Alzheimer’s Disease model by reducing intracellular oxidative stress
[132]. This study together with others indicated that iron oxide nanoparticles-based
nanozymes may help to treat diseases associated with oxidative stress.
4.3 Other Metal Oxides
Many other metal oxides have been explored to mimic natural enzymes [35,
136–149].
4.3.1 Vanadium Oxide as Enzyme Mimics
Vanadium oxide nanomaterials (such as V2O5 and V2O3) have been used to mimic
natural enzymes for biosensing, antibiofouling, cytoprotection, etc. [136–139, 150].
Tremel et al. [136] showed that V2O5 nanowires possessed intrinsic peroxidase
mimicking activity. In a subsequent study, they demonstrated that the V2O5
nanowires mimicked natural vanadium haloperoxidase (Fig. 4.18) [137].
A potential mechanism for the vanadium haloperoxidase mimicking activity was
showed in Fig. 4.18b. Interestingly, it has been demonstrated that the nanozymes
could be used to prevent marine biofouling (Fig. 4.18c–f) [137].
Mugesh, D’Silva, and co-workers [138] showed that V2O5 nanowires could also
mimic GSH peroxidase, an important cellular antioxidation enzyme (Fig. 4.19). By
using intracellular glutathione, the nanozyme could protect cells from both intrinsic
and external oxidative injuries. Moreover, the nanozyme has successfully restored
the ROS balance without disturbing the cell’s own antioxidation defense systems.
As shown in Fig. 4.19, a possible mechanism was proposed. H2O2 could be
adsorbed onto the nanozyme and was then reduced into H2O, leading to the for-
mation of complex 1. Complex 1 then interacted with GSH and formed complex 2,
which would be hydrolyzed into complexes 3 and 4. The reaction of complex 4
with H2O2 would regenerate complex 1. It should be noted that complex 3 could be
converted to GSSG, which was further transformed to GSH by glutathione
reductase (GR) and NADPH [138].
DNA and glucose detection has been reported by using polydopamine-coated
V2O5 nanowires as peroxidase mimics [151]. In another report, V2O3-loaded
mesoporous carbon has been used for colorimetric detection of glucose by using its
peroxidase mimicking activity [139].
4.3.2 Cobalt Oxide as Enzyme Mimics
Several reports have showed that cobalt oxide (especially Co3O4) nanomaterials
could mimic peroxidase, catalase, SOD, etc. [150, 152, 153]. For example, Wang
et al. [152] reported the catalase-like activity of Co3O4 nanoparticles. It was pro-
posed that the catalysis could be mediated via a Co2+ → Co3+ → Co2+ regener-
ation mechanism. In a following study, they demonstrated that the Co3O4
nanoparticles’ catalase mimicking activities could be modulated by controlling the
4.3 Other Metal Oxides 79
Fig. 4.18 a TEM image of V2O5 nanowires. b Proposed mechanism for V2O5 nanowires’
vanadium haloperoxidase mimicking activity. c–f Effect of V2O5 nanowires on marine biofouling.
Digital image of a stainless steel plate (2 × 2 cm) covered with a commercially available paint for
boat hulls without and with V2O5 nanowires. The plates were fixed to a boat hull. c, d Immediately
after fixation, both stainless steel plates (with and without V2O5 nanowires) had clean surfaces.
The boat was kept in seawater (lagoon with tidal water directly connected to the Atlantic Ocean).
After 60 days, the boat was taken from the water. e The painted stainless steel plates without V2O5
nanowires suffered from severe natural biofouling. f Plates with V2O5 nanowires showed a
complete absence of biofouling. Adapted from Ref. [137], Copyright 2012, with permission from
Nature Publishing Group
Fig. 4.19 Proposed molecular mechanism for V2O5 nanowires’ GSH peroxidase mimicking
activity. Reprinted from Ref. [138], Copyright 2014, with permission from Nature Publishing
Group
nanoparticles’ morphologies. The nanozymes exhibited different activities in the

order of nanoplates > nanorods > nanocubes. Moreover, they discovered that the
nanozymes’ activities could be specifically enhanced by Ca2+. Based on this phe-
nomenon, they constructed a biosensor for Ca2+. With the biosensor, spiked Ca2+ in
milk was successfully determined [154].
Zhang, Gu, and co-workers [150] found that the Co3O4 nanoparticles possessed
multiple enzyme-like activities (i.e., catalase-like, peroxidase-like, and SOD-like
activities). For all the three mimics, the Co2+ → Co3+ → Co2+ regeneration
mechanism was involved. Compared with Fe3O4 nanoparticles, Co3O4 nanoparti-
cles exhibited higher enzyme mimicking activities. After establishing the enzyme
mimicking activities, they developed an immunoassay for vascular endothelial
growth factor (VEGF) detection [150].
4.3 Other Metal Oxides 81
4.3.3 Copper Oxide as Enzyme Mimics
Chen’s and others’ groups have reported the enzyme mimicking activities of copper
oxide nanomaterials [147, 155–164]. For instance, Chen et al. demonstrated that
CuO nanoparticles with an average size of 30 nm exhibited peroxidase mimicking
activity [155]. Enzyme kinetics study revealed that the CuO-based nanozyme had
higher affinity toward TMB than natural HRP and several other nanozymes (e.g.,
Fe3O4- and FeS-based peroxidase mimics). By combining the CuO nanozyme with
cholesterol oxidase, they have developed a chemiluminescent biosensor for
cholesterol detection [164]. Glucose and lactate detection was also reported by
using CuO nanoparticles as peroxidase mimics [156].
4.3.4 MoO3, TiO2, MnO2, RuO2 as Enzyme Mimics
Tremel et al. [149] studied the enzyme mimicking activities of MoO3 nanoparticles
and found that they could mimic sulfite oxidase, which catalyzed the oxidation of
sulfite to sulfate. After establishing the sulfite oxidase mimicking activity in vitro,
they further demonstrated that the nanozyme could work in living cells and
recovered the sulfite oxidase activity in sulfite oxidase knockdown cells [149]. In
this case, the intracellular sulfite oxidase activity was chemically inhibited by
sodium tungstate treatment. Since native sulfite oxidase is localized within mito-
chondria, the nanozyme was conjugated with triphenylphosphonium moieties for
mitochondria targeting. When the sulfite oxidase deficient cells were treated with
the nanozymes, their sulfite oxidase activity was restored [149].
Dong et al. fabricated TiO2 nanotube arrays via a potentiostatic anodization
strategy. They further showed that the TiO2 nanotube arrays exhibited
peroxidase-like activity. Sensitive detection of H2O2 was demonstrated by using the
TiO2 nanotube arrays as a working electrode, which showed catalytic activity toward
H2O2 reduction. Moreover, when GOx was immobilized onto the TiO2 nanotube
arrays, an electrochemical sensor for glucose detection was obtained. As shown in
Fig. 4.20, the biosensor exhibited excellent sensitivity and selectivity toward glu-
cose determination. Besides, the biosensor was further used to measure the glucose
concentrations in diabetes patients, showing satisfactory performance [148].
Gao et al. [140] have constructed an electrochemical DNA biosensor by using
the peroxidase mimicking activity of RuO2 nanoparticles. When the RuO2
nanoparticles labeled probe ssDNA was specifically assembled onto an electrode in
a sandwich manner with the capture ssDNA and target ssDNA, the nanoparticles
catalyzed the deposition of polyaniline and produced catalytic electrochemical
signals for detection. Zhang and co-workers demonstrated that MnO2 nanowires
were excellent peroxidase mimics. They then developed an immunoassay to detect
sulfate-reducing bacteria [144].
Fig. 4.20 a Cyclic voltammograms (CVs) obtained at the GOx/TiO2 nanotube array electrode in
0.1 M pH 5.5 PBS without (a) and with (b) 4 mM glucose. b CVs obtained at the TiO2 nanotube
array electrode in 0.1 M pH 5.5 PBS without (a) and with (b) 4 mM glucose. c Amperometric
response obtained at the GOD/TiO2 nanotube array electrode in 0.1 M pH 5.5 PBS upon
successive injection of 0.2 mM glucose for each step at −0.35 V, the inset is the linear fitted
current plot to glucose concentration. d Amperometric response at the GOD/TiO2 nanotube array
electrode in 0.1 M pH 5.5 PBS at −0.35 V with sequential injection of 1 mM glucose (a), 2 mM
fructose (b), 2 mM lactose (c), 2 mM sucrose (d), 2 mM maltose (e), and 1 mM glucose (f).
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Chapter 5
Other Nanomaterials for Nanozymes
Abstract The use of other nanomaterials beyond carbon-based nanomaterials,

metal-based nanomaterials, and metal oxide-based nanomaterials for mimicking
natural enzymes is discussed in this chapter. Prussian blue, metal-organic frame-
works, metal chalcogenides, metal hydroxides, etc., have been selected as repre-
sentative nanomaterials for mimicking peroxidase, superoxide dismutase, catalase,
etc. The catalytic mechanisms are also discussed if they have been elucidated.
Selected examples for in vitro biosensing, in vivo bioanalysis, and therapeutics are
discussed to highlight the broad applications of these nanozymes.

Keywords Nanozymes Artificial enzymes Integrated nanozymes Prussian

blue Metal-organic frameworks Metal chalcogenides Metal hydroxides

Cascade reactions Enzyme mimics Functional nanomaterials
As discussed in Chap. 1, more and more nanomaterials have been explored to

investigate their enzyme mimicking activities [1–60]. To highlight the ever-
growing interests in searching for new nanozymes, in this chapter selected exam-
ples of other nanomaterials for nanozymes are discussed.
5.1 Prussian Blue
Prussian blue, [Fe(III)Fe(II)(CN)6]−, based nanomaterials have been used to mimic

peroxidase, catalase, and SOD [4–6, 61]. In their initial report, Gu et al. found that
the Prussian blue coating could enhance the peroxidase mimicking activity of
γ-Fe2O3 nanoparticles [3]. Later, they showed that Prussian blue nanoparticles
exhibited catalase-like activity at neutral conditions (i.e., pH = 7.4) [4]. The
nanozyme was used for in vivo ultrasound and magnetic-resonance imaging of
overproduced H2O2 in diseased tissues. The nanozyme converted H2O2 into O2 gas
bubbles, which acted as the ultrasound contrast agents. Moreover, due to the
paramagnetic property of the formed O2 gas bubbles, they also acted as T1

94 5 Other Nanomaterials for Nanozymes
Fig. 5.1 a Schematic showing intracellular behaviors of Prussian blue nanoparticles (PBNPs).
b Proposed mechanisms of the multiple enzyme-like activities of PBNPs based on standard redox
potentials of different compounds in the reaction systems. c Reactions involved. Prussian white
(PW, [Fe(II)Fe(II)(CN)6]2−), Berlin green (BG, {Fe(III)3[Fe(III)(CN)6]2[Fe(II)(CN)6]}2−), and
Prussian yellow (PY, [Fe(II)Fe(III)(CN)6]). Reprinted from Ref. [5], Copyright 2016, with
5.1 Prussian Blue 95
magnetic-resonance contrast agents [4]. Using a lipopolysaccharide (LPS)-induced

liver inflammation model, the overproduced H2O2 in living mouse livers was
successfully imaged both by ultrasound and magnetic resonance methods.
Recently, Gu et al. [5] further confirmed that Prussian blue nanoparticles pos-
sessed peroxidase-like, catalase-like, and SOD-like activities (Fig. 5.1). Detailed
experimental results suggested that the peroxidase mimicking activity was domi-
nant at acidic conditions while the catalase mimicking activity was dominant at
high pH conditions. Interestingly, Prussian blue nanoparticles exhibited SOD-like
activities at different pH levels. Due to the mixed valence of Fe2+ and Fe3+, Prussian
blue can be reduced into Prussian white and oxidized into Berlin green or Prussian
yellow. As shown in Fig. 5.1b, the peroxidase-like activity was attributed to the
following mechanism: Prussian blue was first oxidized into Prussian yellow or
Berlin green by H2O2 at acidic pH (Eq. 5.6). Prussian yellow/Berlin green would
then oxidize TMB to into TMBox. Therefore, Prussian yellow/Berlin as the per-
oxidase mimic transferred electrons from TMB to H2O2 to achieve the catalytic
recycle (Eq. 5.7). Since H2O2 could act both as oxidizing and reducing agents, the
presence of the nanozyme would dismutate H2O2 into H2O and O2 via the mech-
anisms shown in Eqs. 5.8–5.11. Similarly, superoxide anion could be converted
into H2O2 and O2 via the mechanisms shown in Eqs. 5.12–5.16. They further
demonstrated that the nanozymes could alleviate inflammation by scavenging ROS
in vivo in lipopolysaccharide-treated mice model [5].
5.2 Metal-Organic Frameworks
Metal-organic frameworks (MOFs) themselves and MOFs loaded with other cata-
lysts have been used to mimic natural enzymes [7–18]. For instance, it has reported
that a well-known Cu2+ and benzene-1,3,5-tricarboxylate ligand-based MOF (i.e.,
HKUST-1) could mimic protease. Similar with natural trypsin, HKUST-1 could
catalyze the hydrolysis of bovine serum albumin (BSA) and casein. Moreover, in
comparison with natural trypsin, the MOF-based nanozyme had higher affinity
toward BSA [9].
For MOFs made from non-redox active metal ions, they could be imparted with
catalytic activities by encapsulating guest catalysts [7, 8, 15]. Usually, one catalyst
was usually encapsulated within MOFs for catalysis [15]. In biological systems,
however, multiple enzymes are usually compartmentalized within subcellular
organelles for cascade catalytic reactions. Such compartmentalization would lead to
an interesting “nanoscale proximity effects,” which could significantly enhance the
coupled reactions via several mechanisms. For example, it could improve the local
concentrations of catalysts and substrates, reduce the diffusion barrier, and stabilize
the catalysts and the unstable intermediates. Inspired by this, several strategies have
been developed to co-encapsulate two catalysts guests within various matrix [7, 8,
62–66].
Wei and co-workers [7, 8] have developed a self-assembly strategy to fabricate

integrated nanozymes (INAzymes) by co-assembling multiple catalytic guests
within MOFs (Fig. 5.2). They first demonstrated that molecular catalysts (e.g.,
hemin) and natural enzymes (e.g., GOx) could be simultaneously confined with
ZIF-8, a MOF made from Zn2+ and 2-methylimidazole ligands, under biocom-
patible reaction conditions (Fig. 5.2a). Moreover, the obtained INAzyme of hemin
and GOx exhibited more than 600 % enhancement of the catalytic activity when
compared with the mixture of hemin@ZIF-8 and GOx@ZIF-8 (Fig. 5.2c). After
establishing that the INAzyme could be used for sensitive and selective detection of
glucose in vitro, they went on to construct an analytical platform by immobilizing
the INAzyme into the channel of a microfluidics chip. When further assisted with
microdialysis, the dynamic changes of brain glucose following ischemia and per-
fusion has been successfully monitored with the platform (Fig. 5.2d, e). Their
strategy was general and applicable to other combination of catalyst guests (such as
hemin/lactate oxidase and hemin/GOx/invertase) [8].
Fig. 5.2 Integrated nanozymes for monitoring the dynamic changes of brain glucose following
ischemia/reperfusion. a Schematic and TEM image of the integrated nanozymes (INAzymes).
b Reactions catalyzed by the INAzymes. c Normalized cascade catalytic activity of the INAzyme
(1), and the mixture of hemin@ZIF-8 and GOx@ZIF-8 (2), showing a more than 600 %
enhancement for the INAzyme when compared with the mixture of hemin@ZIF-8 and
GOx@ZIF-8. d Schematic illustration of the global cerebral ischemia. e Continuously monitoring
the dynamic changes of glucose level in the striatum of a living rat brain following global
ischemia/reperfusion with the INAzyme-based sensing platform. Adapted from Ref. [8], Copyright
2016, with permission from American Chemical Society
5.3 Metal Chalcogenides 97
5.3 Metal Chalcogenides
Numerous studies have showed that metal chalcogenides (such as CuS, MnSe, and
FeSe) could mimic peroxidase [19–34]. For example, Huang et al. prepared NiTe
nanowires via a hydrothermal method. The obtained NiTe nanowires showed
peroxidase mimicking activity, which were better than the commercial NiTe
powders. They further combined the nanozyme with GOx for sensitive and selective
detection of glucose [24].
5.4 Metal Hydroxides
Metal hydroxides, including layered double hydroxides, have been explored to

mimic peroxidase [35–38]. For example, Sun et al. reported the peroxidase-like
activity of CoFe layered double hydroxides and used them for colorimetric
detection of H2O2 and glucose [37]. Tan’s group fabricated an interesting Cu(OH)2
nanocages from amorphous Cu(OH)2 nanoparticles. Catalytic studies revealed that
the nanocages showed higher peroxidase mimicking activities even than that of
natural enzymes (Fig. 5.3) [38].
Fig. 5.3 Cu(OH)2 nanocages

as peroxidase mimics.
Reprinted from Ref. [38],
Copyright 2015, with
permission from American
Chemical Society
Fig. 5.4 a SEM image of polypyrrole/hemin nanocomposites. b Polypyrrole/hemin nanocom-

posites as peroxidase mimics for glucose sensing. Adapted from Ref. [54], Copyright 2014, with
5.5 Miscellaneous
Lots of other materials have been reported to mainly mimic peroxidase [39–60, 67].
For example, magnetic zirconium hexacyanoferrate (II) nanoparticles as peroxidase
mimics were used to label probe ssDNA for electrochemical DNA sensing [56].
Dong et al. [54] developed a facile way to prepare polypyrrole/hemin nanocom-
posites by chemical oxidative polymerization of pyrrole monomer with FeCl3 in the
presence of hemin (Fig. 5.4). The peroxidase-like activity of the nanocomposites
was confirmed by catalyzing the oxidation of TMB and other peroxidase substrates
with H2O2. When the peroxidase mimics were combined with GOx, the selective
and sensitive detection of glucose was achieved [54].
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Chapter 6
Challenges and Perspectives
Abstract The challenges and perspectives in the field of nanozymes are summa-
rized in this chapter, which if fully addressed, will lead to substantial breakthroughs
in the future.

Keywords Nanozymes Artificial enzymes Enzyme mimics Catalytic nano-

materials Nanobiology Nanozymology Functional nanomaterials Biological

catalysts Translational medicine Challenges and perspectives
As has been evidenced in the preceding chapters, highly active research interests
have been devoted to the field of nanozymes owing to their unique characteristics.
Despite the remarkable progress has been made in the field, the nanozyme research is
still in its infancy. Therefore, substantial breakthroughs are expected, which will lead
to the next wave of artificial enzymes for both fundamental science and practical
applications by overcoming the following as well as other challenges [1–4].
(1) Nanozymes with new catalytic properties beyond redox enzyme mimics
The currently developed nanozymes are mainly mimic redox enzymes (such as
peroxidase, oxidase, catalase, and superoxide dismutase) and hydrolytic enzymes
(such as nuclease, esterase) though other enzyme mimics have been reported [5, 6].
Given the fact that there are six major types of natural enzymes, future efforts
should be focused on designing new nanozymes that may mimic their
functionalities.
More, as Professor Dr. Jean-Marie Lehn stated: “the chemist finds inspiration in
the ingenuity of biological events and encouragement in the demonstration that
such high efficiencies, selectivities, and rates can indeed be attained. However,
chemistry is not limited to systems similar to those found in biology, but is free to
create unknown species and to invent novel processes,” researchers are therefore
encouraged to have great ambition to design new nanozymes beyond the natural
ones [7].

104 6 Challenges and Perspectives
(2) Rational design of nanozymes

The successful tackling of the above-mentioned challenge relies on our capa-
bility of rationally designing new nanozymes. To fulfill this goal, one should first
understand the nanozymes’ catalytic mechanisms both experimentally and theo-
retically [8–11]. Advanced characterization techniques, such as in situ (sub)atomic
resolution TEM imaging and synchrotron spectroscopies, are expected to provide
deeper insights. Recent progress in computational chemistry has allowed to design
functional proteins (including enzymes), which should provide invaluable guidance
to nanozyme design [12–14].
Despite the substantial progress in nanotechnology, it remains a great challenge
to prepare uniform (especially the atomically uniform) nanomaterials. On the other
hand, natural enzymes have well-defined amino acid sequences (or nucleic acid
sequences) as well as three-dimensional structures. Therefore, better synthetic
strategies are still needed to prepare highly efficient nanozymes with defined sizes
and structures even one may rationally design them in the future.
For practical applications, large-scale synthesis of nanozymes is also expected in
the near future. Industrial standards (and other necessary standards) should be
established for both nanozymes’ synthesis and characterization.
(3) Regulation of nanozymes’ activities
In biological systems, enzymes’ activities are highly regulated. Inspired by this
interesting phenomenon, numerous methods have been developed to tune the
nanozymes’ activities [1, 15–17]. In nature, an enzyme’s activity could be tuned by
regulating its expression and composition at genetic level and by controlling its
surrounding environment and its interaction with specific ligands. All of these
approaches should be exploited to regulate the nanozymes’ activities. For instance, in
a cell, multiple enzymes usually work together within confined compartments for
synergetic cascade reactions. Recent studies demonstrated that nanozymes could also
work cooperatively by co-assembling them together within confined spaces [15].
To obtain highly efficient nanozymes, the catalytic activities from both the core
and the surface coating should be exploited. For the nanozymes with catalytically
active core, the surface coating may shield their activities. Therefore, suitable
coatings should be adopted. On the other hand, some nanozymes’ cores only acts as
the supporting material. In this case, highly efficient nanozymes could be obtained
by exploring the synergistic activities of both the inorganic core and the surface
coatings. Also by introducing chiral cores or surface coatings, nanozymes with
chiral selectivities would be produced [18].
(4) Biosafety of nanozymes
Both the dynamic and final fates of nanozymes should be systematically studied
at different levels to address the potential toxicity concerns [19–24]. For example,
previous studies showed that the nanoparticles’ surface properties would affect their
interaction with cell membrane and their subcellular localizations [25–27].
Biodistribution studies suggested that the nanozymes without targeting molecules
6 Challenges and Perspectives 105
would accumulate mainly in liver, spleen, and lung [28, 29]. Other factors, like
nanoparticles’ aspect ratio, may also affect their biodistributions [30].
Encouragingly, several studies showed that a few regulatory agencies approved
that nanomaterials (such as Resovist (Ferucarbotran), i.e., the commercial available
superparamagnetic iron oxide nanoparticles) have been used as nanozymes [31].
However, for most of the currently developed nanozymes, both their acute and
long-term biosafety should be evaluated before they could be applied in clinics.
(5) Translational promise of nanozymes
As closely related with the biosafety issues discussed above, the potential
applications of the nanozymes in translational medicine and clinics remain largely
unexplored. Though the therapeutic effects of several nanozymes have been
investigated, much more efforts are needed to translate the encouraging lab (and
even pre-clinical) results into clinics to benefit patients and the society [32].
(6) Beyond the catalysis
Though it has suggested that redox active nanozymes showed promising neu-
roprotection and antioxidation effects via catalytic mechanisms, their applications
definitely could be extended to other areas [33]. For instance, they could be used to
modulate enzymes’ activities in vitro [34]. Overall, we believe that nanozymes will
be widely adopted not only as highly efficient biocatalysts but also as versatile tools
in nanobiology, an interfaced area of nanotechnology, biology, biomedicine, etc.
We and others have also speculated that nanozymes may even play a role in the
early stages of life [2, 35].
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Appendix
See Tables A.1, A.2 and A.3.
Table A.1 H2O2 detection with peroxidase mimics

Nanozymes Meth Linear range LOD Comments Ref.
Fe3O4 MNPs Color 5–100 μM 3 μM Substrate: ABTS [1]
Fe3O4 MNPs Color 0.5–150.0 μM 0.25 μM Substrate: DPD [2]
H2O2 in rainwater,
honey, and milk was
tested
Fe3O4 MNPs Color 1–100 μM 0.5 μM Substrate: TMB [3]
Fe3O4 was
encapsulated in
mesoporous silica
Fe3O4 graphene Color 1–50 μM 0.32 μM Substrate: TMB [4]
oxide composites
Fe-substituted SBA– Color 0.4–15 μM 0.2 μM Substrate: TMB [5]
15 microparticles
Iron phosphate Color 10–50 μM 10 nM Substrate: TMB [6]
microflowers
[Fe(III) Color 0.1–5 mM 10 μM Substrate: TMB [7]
(biuret-amide)] on
mesoporous silica
FeTe nanorods Color 0.1–5 μM 55 nM Substrate: ABTS [8]
Fe(III)-based Color 1–50 μM 0.4 μM Substrate: TMB [9]
coordination
polymer
Fe3O4 Color 5–80 μM 1.07 μM Substrate: TMB [10]
nanocomposites Fe3O4 was
functionalized by
5,10,15,20-Tetrakis
(4-carboxyphenyl)-
porphyrin
(continued)

SpringerBriefs in Molecular Science, DOI 10.1007/978-3-662-53068-9
110 Appendix
Table A.1 (continued)

Protein-Fe3O4 and Color 0.5–200 μM 0.2 μM Substrate: TMB [11]
glucose oxidase
nanocomposites
GOx/Fe3O4/GO Color 0.1–100 μM 0.04 μM Substrate: DPD [12]
magnetic
nanocomposite
Iron(III) hydrogen Color 57.4–525.8 μM 1 μM Substrate: TMB [13]
phosphate hydrate
crystals
MIL-53(Fe) Color 0.95–19 μM 0.13 μM Substrate: TMB [14]
MIL-53(Fe): a metal–
organic framework
CuO NPs Color 0.01–1mM N/A Substrate: 4-AAP and [15]
phenol
AuNPs Color 18–1100 μM 4 μM Substrate: TMB [16]
Cysteamine was the
ligand for AuNPs
AuNC@BSA Color 0.5–20 μM 20 nM Substrate: TMB [17]
Au@Pt core/shell Color 45–1000 μM 45 μM Substrate: OPD [18]
nanorods
Nickel telluride Color 0.1–0.5 μM 25 nM Substrate: ABTS [19]
nanowires
Graphene oxide Color 0.05–100 μM 50 nM Substrate: TMB [20]
Hemin–graphene Color 0.05–500 μM 20 nM Substrate: TMB [21]
hybrid nanosheets
Carbon nanodots Color 1–100 μM 0.2 μM Substrate: TMB [22]
Carbon nitride dots Color 1–100 μM 0.4 μM Substrate: TMB [23]
Tungsten carbide Color 0.2–80 μM 60 nM Substrate: TMB [24]
nanorods
CoFe LDH Color 1–20 μM 0.4 μM Substrate: TMB [25]
nanoplates
CoxFe3–xO4 Color. 1–60 μM 0.36 μM Substrate: TMB [26]
nanocubes
Porphyrin Color 1–75 μM 0.4 μM Substrate: TMB [27]
functionalized
Co3O4
nanostructures
Carboxyl Color 1–8 μM 0.4 μM Substrate: TMB [28]
functionalized
mesoporous polymer
PtPd nanodendrites Color 0.5–150 μM 0.1 μM Substrate: TMB [29]
on graphene
nanosheets
(PtPdNDs/GNs)
(continued)
Appendix 111

Pt-DNA complexes Color 0.979–17.6 mM 0.392 mM Substrate: TMB [30]
3.92 μM was detected
with PVDF
membrane
Manganese selenide Color 0.17–10 μM 0.085 μM Substrate: TMB [31]
nanoparticles
Prussian blue Color 0.05–50 μM 0.031 μM Substrate: ABTS [32]
nanoparticles
MWCNTs-Prussian Color 1 μM–1.5 mM 100 nM Substrate: TMB [33]
blue nanoparticles Carbon nanotubes
were filled with
Prussian blue
nanoparticles
Polypyrrole Color 5–100 μM Substrate: TMB [34]
nanoparticles PPy has been
successfully
employed to
quantitatively monitor
the H2O2 generated
by macrophages
Polyoxometalate Color 1–20 μM 0.4 μM Substrate: TMB [35]
Polyoxometalate Color 0.134–67 μM 0.134 μM Substrate: TMB [36]
Fe3O4 MNPs Fluor 10–200 nM 5.8 nM Substrate: Rhodamine [37]
B
Fluorescence of
Rhodamine B was
quenched
BiFeO3 NPs Fluor 20 nM–20 μM 4.5 nM Substrate: BA [38]
Oxidation of BA gave
fluorescence
H2O2 in rainwater
was tested
Fe3O4 MNPs Fluor 0.18–900 μM 0.18 μM Fluorescence of [39]
CdTe QD was
quenched
Fe3O4 MNPs Fluor 0.04–8 μM 0.008 μM Substrate: BA [40]
fluorescence
cupric oxide Fluor 5–200 μM 0.34 μM Substrate: terephthalic [41]
nanoparticles acid
Terephthalic acid was
oxidized by hydroxyl
radical to form a
highly fluorescent
product
(continued)
112 Appendix

Fe(III)–TAML CL 0.06–1 μM 0.05 μM [42]
activator
CoFe2O4 NPs CL 0.1–4 μM 0.02 μM CoFe2O4 NPs form [43]
complexes with
beta-CD
CoFe2O4 NPs CL 0.1–10 μM 10 nM H2O2 in natural water [44]
was tested
CoFe2O4 NPs with CL 1 nM–4 μM 0.5 nM CoFe2O4 NPs was [45]
chitosan coating coated with chitosan
H2O2 in natural water
was tested
Fe3O4 MNPs E-chem 4.2–800 μM 1.4 μM [46]
Fe3O4 E-chem 1.2–3500 μM 1.2 μM H2O2 in disinfected [47]
microspheres-AgNP FBS samples was
hybrids tested
Fe3O4 MNPs E-chem 0–16 nM 1.6 nM Fe3O4 was loaded on [48]
CNT
Fe3O4 MNPs E-chem 1–10 mM N/A Fe3O4 was entrapped [49]
in mesoporous carbon
foam, and the
composite was used
to construct a carbon
paste electrode
Not a linear response
Fe3O4 MNPs E-chem 20–6250 μM 2.5 μM Fe3O4 MNPs and [50]
PDDA–graphene
formed multilayer via
layer-by-layer
assembly
H2O2 in toothpaste
was tested
Fe3O4 E-chem 1–700 μM 1 μM H2O2 in Walgreens [51]
nanofilms on TiN antiseptic/oral
substrate debriding agent, Crest
whitening mouthwash
solution, Diet coke,
and Gatorade was
tested
Fe3O4 MNPs E-chem 0.2–2 mM 0.01 mM [52]
Fe3O4 MNPs E-chem 0.1–6 mM 3.2 μM Fe3O4 was on reduced [53]
graphene oxide
Fe2O3 NPs E-chem 20–140 μM 11 μM [54]
Fe2O3 NPs E-chem 20–300 μM 7 μM Fe2O3 was modified [54]
with Prussian blue.
Iron oxide NPs/CNT E-chem 0.099–6.54 mM 53.6 μM [55]
(continued)
Appendix 113

Fe3O4/self-reduced E-chem 0.001–20 mM 0.17 μM Extracellular H2O2 [56]
graphene released from HeLa
nanocomposites cells stimulated by
CdTe quantum dots
(QDs) was established
by this approach
FeS nanosheet E-chem 0.5–150 μM 92 nM [57]
FeS needle E-chem 5–140 μM 4.3 μM [58]
FeSe NPs E-chem 5–100 μM 3.0 μM [58]
FeS E-chem 10–130 μM 4.03 μM [59]
Co3O4 NPs E-chem 0.05–25 mM 0.01 mM [60]
Hemin–graphene E-chem 0.5–400 μM 0.2 μM [21]
hybrid nanosheets
Layered double E-chem 1–240 μM 0.3 μM [61]
hydroxide–hemin
nanocomposite
Helical CNT E-chem 0.5–115 μM 0.12 μM [62]
LDH nanoflakes E-chem 12–254 μM 2.3 μM [63]
Calcined LDH E-chem 1–100 μM 0.5 μM [64]
CdS E-chem 1–1900 μM 0.28 μM [65]
Table A.2 Targets detection combining oxidases and peroxidase mimics

Glucose
Fe3O4 MNPs Color 50–1000 μM 30 μM Substrate: ABTS [1]
Selectivity against
sugars: fructose,
lactose, and maltose
Fe3O4 MNPs with Color 39–100 μM 30 μM Substrate: ABTS [66]
PDDA coating GOx was
electrostatically
assembled onto the
Fe3O4@PDDA
Glucose in serum
samples was tested
Compared with
glucometer
Selectivity against
sugars: galactose,
lactose, mannose,
maltose, arabinose,
cellobiose, raffinose,
and xylose
(continued)
114 Appendix

Fe3O4 MNPs Color 30–1000 μM 3 μM Substrate: TMB [3]
Fe3O4 was
encapsulated in
mesoporous silica with
GOx
Showing the recycle
capability
Comparison between
free MNPs versus
encapsulated MNPs
Fe3O4 GO Color 2–200 μM 0.74 μM Substrate: TMB [4]
composites Glucose in urine was
tested
Fe3O4 Color 5–25 μM 2.21 μM Substrate: TMB [10]
nanocomposites Fe3O4 was
functionalized by
5,10,15,20-Tetrakis
(4-carboxyphenyl)-
porphyrin
Protein-Fe3O4 and Color 3–1000 μM 1.0 μM Substrate: TMB [11]
glucose oxidase
nanocomposites
γ-Fe2O3 Color 1–80 μM 0.21 μM Substrate: TMB [67]
nanoparticles Glucose in blood and
urine was tested
GOx/Fe3O4/GO Color 0.5–600 μM 0.2 μM Substrate: DPD [12]
magnetic
nanocomposite
Graphite-like carbon Color 5–100 μM 0.1 μM Substrate: TMB [68]
nitrides Glucose in serum was
tested
Iron oxide NPs Color 31.2–250 μM 8.5 μM Substrate: ABTS [69]
Iron oxide NPs was
coated with glycine
More robust than HRP
towards NaN3
inhibition
Iron oxide NPs was
coated with heparin
More robust than HRP
towards NaN3
inhibition
Iron oxide NPs was
coated with APTES
and MPTES
(continued)
Appendix 115

ZnFe2O4 Color 1.25–18.75 μM 0.3 μM Substrate: TMB [71]
Glucose in urine was
tested
[Fe(III) Color 20–300 μM 10 μM Substrate: TMB [7]
(biuret-amide)] on Glucose in mice blood
mesoporous silica plasma was tested
FeTe nanorods Color 1–100 μM 0.38 μM Substrate: ABTS [8]
Glucose in spiked
blood was tested
Fe(III)-based Color 2–20 μM 1 μM Substrate: TMB [9]
coordination polymer Glucose in serum was
tested
Mesoporous Fe2O3– Color 0.5–10 μM 0.5 μM Substrate: TMB [72]
graphene Glucose in serum was
nanostructures tested
CuO NPs Color 0.1–8 mM N/A Substrate: 4-AAP and [15]
phenol
V2O5 nanowires and Color 0–10 μM 0.5 μM Substrate: ABTS [73]
Gold nanoparticles
nanocomposite
AuNPs Color 2.0–200 μM 0.5 μM Substrate: TMB [16]
Cysteamine was the
ligand for AuNPs
nanorods
Nickel telluride Color 1–50 μM 0.42 μM Substrate: ABTS [19]
nanowires
Manganese selenide Color 8–50 μM 1.6 μM Substrate: TMB [31]
nanoparticles
Graphene oxide Color 1–20 μM 1 μM Substrate: TMB [20]
Glucose in blood and
fruit juice was tested
Graphene oxide Color 2.5–5 mM 0.5 μM Substrate: TMB [74]
Graphene oxide was
functionalized by
chitosan
Hemin–graphene Color 0.05–500 μM 30 nM Substrate: TMB [21]
hybrid nanosheets
Carbon nanodots Color 1–500 μM 1 μM Substrate: TMB [22]
Glucose in serum was
tested
Carbon nitride dots Color 1–5 μM 0.5 μM Substrate: TMB [23]
(continued)
116 Appendix

MWCNTs–Prussian Color 1 μM–1 mM 200 nM Substrate: TMB [33]
blue nanoparticles Carbon nanotubes
were filled with
Prussian blue
nanoparticles
CoFe LDH Color 1–10 mM 0.6 μM Substrate: TMB [25]
nanoplates
CoxFe3-xO4 Color 8–90 μM 2.47 μM Substrate: TMB [26]
nanocubes
MoS2 Color 5–150 μM 1.2 μM Substrate: TMB [75]
nanosheets Glucose in serum was
tested
Tungsten disulfide Color 5–300 μM 2.9 μM Substrate: TMB [76]
nanosheets Glucose in serum of
normal persons and
diabetes persons was
tested
Prussian blue Color 0.1–50 μM 0.03 μM Substrate: ABTS [32]
nanoparticles
Fe3O4 MNPs Fluor 1.6–160 μM 1.0 μM Fluorescence of [39]
CdTe QD was
quenched
tested
Fe3O4 MNPs Fluor 0.05–10 μM 0.025 μM Substrate: benzoic acid [40]
fluorescence
tested
Fe3O4 MNPs with Fluor 3–9 μM 3 μM GOx was [77]
PDDA coating electrostatically
assembled onto the
Fe3O4@PDDA
Oxidation of AU gave
fluorescence
tested
Selectivity against
sugars: arabinose,
cellobiose, galactose,
lactose, maltose,
raffinose, and xylose
BiFeO3 NPs Fluor 1–100 μM 0.5 μM Oxidation of BA gave [38]
fluorescence
tested
CoFe2O4 NPs CL 0.1–10 μM 0.024 μM Other sugars [44]
(continued)
Appendix 117

CoFe2O4 NPs CL 0.05–10 μM 10 nM CoFe2O4 NPs were [45]
coated with chitosan
tested
Hemin–graphene E-chem 0.5–400 μM 0.3 μM [21]
hybrid nanosheets
Fe3O4 MNPs E-chem 6–2200 μM 6 μM Glucose in serum was [78]
tested
Compared with
clinical analyzer
Nafion for high
selectivity against AA,
UA, sucrose, and
lactose
Fe3O4 MNPs E-chem 0.5–10 mM 0.2 mM Fe3O4 was [49]
encapsulated in
mesoporous carbon
with GOx, and the
composite was used to
construct a carbon
paste electrode
Comparison between
free MNPs vs
encapsulated MNPs
Fe3O4–enzyme– E-chem 0.5 μM– 0.3 μM Glucose in serum was [79]
polypyrrole 34 mM tested
nanoparticles
Ascorbic acid
MIL-53(Fe) Color 28.6–190.5 μM 15 μM Substrate: TMB [14]
MIL-53(Fe): A Metal–
Organic Framework
Dopamine
CoxFe3-xO4 Color 0.6–8 μM 0.13 μM Substrate: TMB [80]
nanoparticles Dopamine in serum
was tested
Thrombin
Ag/Pt bimetallic Color 1–50 nM 2.6 nM Ag/Pt bimetallic [81]
nanoclusters nanoclusters was
produced through a
DNA-templated
method
Glutathione
Fe-MIL-88NH2 Color 1–100 μM 0.45 μM Substrate: TMB [82]
MOF
Cysteine
MOF
(continued)
118 Appendix

Homocysteine
MOF
Choline
Fe3O4 Fluor 20–100 μM 20 μM Choline oxidase was [77]
MNPs with PDDA electrostatically
coating assembled onto the
Fe3O4@PDDA
fluorescence
Fe3O4 MNPs E-chem 1 nM–10 mM 0.1 nM Fe3O4 and choline [83]
(log) oxidase were
immobilized together
on electrode
Selectivity against AA
and UA
Platinum Color 6–400 μM 2.5 μM Substrate: N-ethyl-N- [84]
nanoparticles (3-sulfopropyl)-
3-methylaniline
sodium salt and
4-amino-antipyrine
Acetylcholine
Fe3O4 Color 100 nM– 39 nM Substrate: TMB [85]
nanospheres/reduced 10 mM
graphene oxide
Platinum Color 10–200 μM 2.84 μM Substrate: N-ethyl-N- [84]
nanoparticles (3-sulfopropyl)-3-
methylaniline sodium
salt and
4-amino-antipyrine
Glutathione
Carbon nanodots Color 0–7 μM 0.3 μM Substrate: TMB [86]
Cholesterol
Fe3O4 MNPs Color 10–250 μM 5 μM Substrate: TMB [3]
Fe3O4 was
encapsulated in
mesoporous silica with
cholesterol oxidase
Showing the recycle
capability
Comparison between
free MNPs versus
encapsulated MNPs
(continued)
Appendix 119

nanorods
Galactose
Fe3O4 MNPs Color 10–200 mg/L 5 mg/L Substrate: ABTS [87]
Galactose in dried
blood samples from
normal persons and
patients was tested
Plates were used for
sensing
Fe3O4 MNPs with Fluor 2–80 μM 2 μM Galactose oxidase was [77]
PDDA coating electrostatically
assembled onto the
Fe3O4@PDDA
fluorescence
Melamine
Bare gold Color 1–800 nM 0.2 nM Substrate: TMB [88]
nanoparticles
Kanamycin
Gold nanoparticles Color 1–100 nM 4.52 nM Substrate: TMB [89]
Gold nanoparticles
were modified by
kanamycin aptamer
Xanthine
AuNC@BSA Color 1–200 μM 0.5 μM Substrate: TMB [17]
Xanthine in serum and
urine samples was
tested
Mercury(II)
Ag nanoparticles Color 0.5–800 nM 0.125 nM Substrate: TMB [90]
Mercury(II) in blood
and wastewater was
tested
carbon nanodots Color 0–0.46 μM 23 nM Substrate: TMB [91]
Platinum Color 0.01–4 nM 8.5 pM Substrate: TMB [92]
Nanoparticle
Calcium ion
Co3O4 Nanomaterials E-chem 0.1–1 mM 4 μM The calcium ion in a [93]
milk sample was
tested
120 Appendix
Table A.3 Nanozyme as peroxidase mimics for immunoassay

Nanozyme Target Format Comments Ref.
Fe3O4 NPs with PreS1 Antigen–down [94]
dextran coating immunoassay
TnI Capture-detection
sandwich
immunoassay
Fe3O4 NPs with Mouse IgG Antigen-down [95]
chitosan coating immunoassay
CEA Capture-detection
sandwich
immunoassay
CEA Sandwich
immunoassay
Fe2O3 NPs with IgG Antigen-down [96]
Prussian blue immunoassay
coating
Ferric nanocore Avidin antigen-down Avidin-biotin interaction [97]
residing in immunoassay
ferritin Nitrated Sandwich
human immunoassay
ceruloplasmin
Fe(1–x)MnxFe2O4 Mouse IgG Antigen-down Both direct and indirect assay [98]
NPs with immunoassay
PMIDA coating
MnFe2O4 NPs Sticholysin II Antigen-down [99]
with citric acid immunoassay
coating
Fe-TAML human IgG Antigen-down Fe-TAML was encapsulated [100]
immunoassay Inside Mesoporous Silica
Nanoparticles
Co3O4 vascular Antigen-down [101]
nanoparticles endothelial immunoassay
growth factor
Platinum cytokeratin Sandwich [102]
nanoparticles 19 fragments immunoassay
Platinum folate Antigen-down [103]
Nanoparticles on receptors immunoassay
Graphene Oxide
Gold respiratory Sandwich The peroxidase-like activity [104]
nanoparticles– syncytial immunoassay of gold
graphene oxide virus Nanoparticles–graphene
hybrids oxide hybrids could be
enhanced by mercury(II)
Rod-shaped human IgG Antigen-down The detection limit can be as [105]
Au@PtCu immunoassay low as 90 pg/mL
(continued)
Appendix 121

Nanozyme Target Format Comments Ref.
Au@Pt nanorods mouse IL-2 Sandwich [106]
with PSS coating immunoassay
Graphene oxide PSA Sandwich Clinical samples were tested [107]
immunoassay
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124 Appendix
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Appendix 125
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Nanozymes Next Wave of Artificial Enzymes

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SPRINGER BRIEFS IN MOLECULAR SCIENCE

Nanozymes: Next Wave

Wenjing Guo Hui Wei

ISSN 2191-5407 ISSN 2191-5415 (electronic)

Library of Congress Control Number: 2016946947

© The Author(s) 2016

Printed on acid-free paper

This Springer imprint is published by Springer Nature

Nanjing Hui Wei

3.2 Metal Nanomaterials with Intrinsic Enzyme Mimicking

PSA Prostate-speciﬁc antigen

© The Author(s) 2016 1

To tackle these drawbacks, intensive efforts have been devoted to developing

Table 1.1 Comparison between nanozymes and othersa, b

31. Prins, L. J. (2015). Emergence of complex chemistry on an organic monolayer. Accounts of

Abstract Carbon-based nanomaterials, such as fullerene, graphene, carbon nan-

Carbon-based nanomaterials, such as fullerene, carbon nanotubes (CNTs), gra-

2.1 Fullerene and Derivatives

© The Author(s) 2016 7

2.1.1 Fullerene and Derivatives as Nuclease Mimics

Nuclease catalyzes the cleavage of phosphodiester bond between two nucleotides in

2.1.2 Fullerene and Derivatives as SOD Mimics

(a) Fullerenes as SOD mimics: in vitro activities and mechanisms

(b) Fullerenes as SOD mimics: in vivo applications

2.1.3 Fullerene Derivatives as Peroxidase Mimics

2.2 Graphene and Derivatives

2.2.1 Graphene and Its Derivatives as Peroxidase Mimics

The peroxidase-mimicking activities have been studied mainly with graphene

Fig. 2.5 Deciphering

Since HO∙ was involved in peroxidase-mimicking activity of GQDs, they have

2.2.2 Decorated Graphene (or Its Derivatives) as Peroxidase

Fig. 2.6 Hemin-decorated rGO as peroxidase mimic. a Schematic illustration of peroxidase-like

It is known that single-stranded DNA (ssDNA) and double-stranded DNA

Fig. 2.7 Hemin-decorated rGO as peroxidase mimic for single-nucleotide polymorphisms.

the developed immunoassay, as low as 5.0 pg/mL of aflatoxin B1 was successfully

2.3 Carbon Nanotubes

2.3.1 Carbon Nanotubes as Peroxidase Mimics

Qu and coworkers reported the peroxidase-mimicking activity of single-walled

2.3.2 Carbon Nanotubes as Other Enzyme Mimics

2.4 Other Carbon-Based Nanomaterials

Other carbon-based nanomaterials (such as carbon nanohorn, carbon nanodots,

2.4.1 Other Carbon Nanomaterials as Peroxidase Mimics

Several groups studied the peroxidase-mimicking activities of carbon nanohorns,

2.4.2 Other Carbon Nanomaterials as SOD Mimics

The SOD-mimicking activities of nitrogen-doped carbon nanodots have been

Fig. 2.15 Carbon nanomaterials as peroxidase mimics. a Single-walled carbon nanohorns as

3.1 Metal Nanomaterials with Catalytic

© The Author(s) 2016 31

3.1.1 AuNPs Protected by Alkanethiol with

(a) Alkanethiol-protected AuNPs as RNase mimics: activities and mechanisms

Fig. 3.1 Alkanethiol-protected AuNPs as metallonuclease mimics. a Transphosphorylation

As shown in Fig. 3.2, in comparison with the uncatalyzed transphosphorylation

Fig. 3.2 a Comparison of catalyzed and uncatalyzed transphosphorylation of HPNPP. b Structure

Eelectrostatic / ðQ1 Q2 Þ=ðe r1;2 Þ ð3:1Þ

To test this hypothesis, AuNP-based nanozymes with different polarities have

Fig. 3.3 a AuNP-based nanozymes with different polarities. b Transphosphorylation of HPNPP

3.1.2 AuNPs Protected by Alkanethiol with Non-covalently

supramolecular regulation mechanism, the activities of the nanozymes were mod-

3.1.3 AuNPs Protected by Thiolated Biomolecules

Other AuNPs-based nanozymes have been developed by assembling thiolated