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Enzyme-Like Activity of Nanomaterials

ab b c b c
Weiwei He , Wayne Wamer , Qingsu Xia , Jun-jie Yin & Peter P. Fu
a
Key Laboratory of Micro-Nano Materials for Energy Storage and
Conversion of Henan Province, Institute of Surface Micro and Nano
Materials, Xuchang University, Xuchang, Henan, China
b
Center for Food Safety and Applied Nutrition, U.S. Food and Drug
Administration, College Park, Maryland, USA
c
National Center for Toxicological Research, Food and Drug
Administration, Jefferson, Arizona, USA
Published online: 29 May 2014.

To cite this article: Weiwei He, Wayne Wamer, Qingsu Xia, Jun-jie Yin & Peter P. Fu (2014) Enzyme-
Like Activity of Nanomaterials, Journal of Environmental Science and Health, Part C: Environmental
Carcinogenesis and Ecotoxicology Reviews, 32:2, 186-211, DOI: 10.1080/10590501.2014.907462

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 Journal of Environmental Science and Health, Part C, 32:186–211, 2014
Copyright C Taylor & Francis Group, LLC
ISSN: 1059-0501 print / 1532-4095 online
DOI: 10.1080/10590501.2014.907462

Enzyme-Like Activity
of Nanomaterials
Weiwei He,1,2 Wayne Wamer,2 Qingsu Xia,3
Downloaded by [York University Libraries] at 13:54 17 June 2014

Jun-jie Yin,2 and Peter P. Fu3

1
Key Laboratory of Micro-Nano Materials for Energy Storage and Conversion of
Henan Province, Institute of Surface Micro and Nano Materials, Xuchang University,
Xuchang, Henan, China
2
Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration,
College Park, Maryland, USA
3
National Center for Toxicological Research, Food and Drug Administration, Jefferson,
Arizona, USA

Due to possessing an extremely small size and a large surface area per unit of vol-
ume, nanomaterials have specific characteristic physical, chemical, photochemical, and
biological properties that are very useful in many new applications. Nanoparticles’
catalytic activity and intrinsic ability in generating or scavenging reactive oxygen
species in general can be used to mimic the catalytic activity of natural enzymes. Many
nanoparticles with enzyme-like activities have been found, potentially capable of be-
ing applied for commercial uses, such as in biosensors, pharmaceutical processes, and
the food industry. To date, a variety of nanoparticles, especially those formed from no-
ble metals, have been determined to possess oxidase-like, peroxidase-like, catalase-like,
and/or superoxide dismutase-like activity. The ability of nanoparticles to mimic enzy-
matic activity, especially peroxidase mimics, can be used in a variety of applications,
such as detection of glucose in biological samples and waste water treatment. To study
the enzyme-like activity of nanoparticles, the electron spin resonance method repre-
sents a critically important and convenient analytical approach for zero-time detection
of the reactive substrates and products as well as for mechanism determination.

Keywords: Nanomaterial; peroxidase-like activity, oxidase-like activity; catalase-like

activity, superoxide dismutase-like activity; electron spin resonance

INTRODUCTION
Nanomaterials are chemical entities at least one dimension smaller than
100 nm [1]. With such an extremely small size and large surface area per unit

Address correspondence to Jun-jie Yin, Center for Food Safety and Applied Nutrition,
U.S. Food and Drug Administration, 5100 Paint Branch Parkway, College Park, MD
20740, USA. E-mail: junjie.yin@fda.hhs.gov
Color versions of one or more of the figures in the article can be found online at
www.tandfonline.com/lesc.
186
 Enzyme-Like Activity of Nanomaterials 187
of volume, nanomaterials have characteristic physical, chemical, photochem-
ical, and biological properties that are very different from those of the same
material in bulk form. These unique chemical and biological properties are
useful in many new applications, producing numerous new commercial prod-
ucts used in industry, agriculture, business, medicine, clothing, cosmetics, and
food in our daily lives [1–11].
Natural enzymes, most of which are proteins, exhibit highly efficient cat-
alytic activity, high substrate specificity, and high selectivity in biological re-
actions [12–14]. However, natural enzymes have intrinsic drawbacks for com-
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mercial applications. Their catalytic activity and stability can be affected by

environmental conditions, denaturation, and digestion. They are frequently
difficult to prepare and purify. Partly due to these disadvantages, many man-
made enzymes (artificial enzyme mimetics) are synthesized. Compared with
natural enzymes, the advantages of artificial enzymes are stability, low cost,
and ease of preparation; the disadvantages of enzyme mimetics are that the
catalytic efficiency, specificity, and selectivity are relatively low [14]. To date,
many enzyme mimetics have been prepared and have activities analogous to
cytochrome P450, serine protease, dioxygenase, phosphodiesterase, lipase, acy-
lase, ligase, hydrolase, aldolase, superoxide dismutase, and nitrile hydratases
[15, 16].
One biological characteristic of nanomaterials is enzyme-like activity.
Nanoparticles’ catalytic activity and intrinsic ability in generating or scaveng-
ing reactive oxygen species (ROS) in general can be used to mimic the catalytic
activity of natural enzymes. To date, many nanoparticles with enzyme-like
activities have been found, potentially capable of being applied for commer-
cial uses, such as in biosensors, pharmaceutical processes, and the food indus-
try [14–18]. For example, platinum (Pt) and palladium (Pd) nanoparticles can
scavenge superoxide anion radicals effectively and thus can be used as super-
oxide dismutases (SOD) mimics [14, 19, 20]. Gold (Au) nanoparticles that cat-
alyze the decomposition of H2 O2 to oxygen can be used to mimic catalase [14,
19, 20]. These findings illustrate the particular importance of exploring the
use of nanoparticles as new enzyme mimetics and related applications. Conse-
quently, nanoparticles have been recognized as a promising candidate as en-
zyme mimetics. Compared with natural enzymes, nanoparticles’ enzyme-like
activity nanoparticles has the advantages of facile synthesis in low-cost, tun-
able catalytic activities, and high stability against stringent conditions [14, 20,
21]. To date, a variety of nanoparticles, especially those formed from noble met-
als, have been determined to possess oxidase-like, peroxidase-like, catalase-
like, and/or SOD-like activity [14, 19, 20]. The ability of nanoparticles to mimic
enzymatic activity, especially peroxidase mimics, can be used in a variety of
applications. In this review, we briefly address the enzyme-like activities of
different nanoparticles, and summarize the working principles most employed
in bio-detection using nanoparticles in place of natural enzymes, such as for
 188 W. He et al.

enzyme-free immunoassays and biosensors for colorimetric detection of bio-

logical molecules. In addition, we discuss the benefits of using electron spin
resonance (ESR) methodology as an important analytical approach for zero-
time detection of free radicals, superoxide anion radicals, hydrogen peroxide,
molecular oxygen, and hydroxyl radicals [2, 10–12, 22–29]. In this review, we
illustrate how we applied the ESR method to study each type of the enzyme-
like activities of nanoparticles.
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ENZYME-LIKE ACTIVITY OF NANOPARTICLES

Peroxidase
Peroxidases are a family of enzymes that catalyze biological reactions, in
which peroxide, such as hydrogen peroxide and lipid peroxide, is reduced and
at the same time a redox substrate is oxidized as an electron donor.

peroxidase
2 AH + H2 O2 −−−−−
−→ 2 A + 2 H2 O
peroxidase
2 AH + ROOH −−−−−
−→ 2 A + ROH + H2 O

Hydrogen peroxide (H2 O2 ) is formed in living organisms. Overproduction

of H2 O2 is associated with the development of a variety of inflammatory-type
diseases, including atherosclerosis, chronic obstructive pulmonary disease, and
hepatitis [5, 30]. As shown in the equation, peroxidase can catalytically scav-
enge hydrogen peroxide to form water.
Most peroxidases, such as cytochrome c peroxidase, exhibit high sub-
strate specificity. On the other hand, horseradish peroxidase has very low
substrate specificity. It is commonly used as a peroxidase standard for the
study of peroxidation reactions. In 2007, Gao and colleagues [31] first re-
ported that a nanoparticle, Fe3 O4 magnetic nanoparticles (nano-Fe3 O4 ), pos-
sessed intrinsic peroxidase-like activity. Based on this finding, they proposed
that nanoparticles possessing peroxidase-like activity can be used for many ap-
plications. They demonstrated that nano-Fe3 O4 can be used in a colorimetric
method for detection of glucose [31]. After this finding, many other nanopar-
ticles, including fullerene, single-wall carbon nanotubes and graphene oxides
[30, 32–36], iron oxide magnetic nanoparticles [16, 37–42], positively charged
gold nanoparticles [25, 43], platinum nanoparticles [23, 44], bimetallic alloy
nanoparticles [12, 24, 28, 45, 46], and CeO2 and Co3 O4 nanoparticles [13, 47],
have been determined to exhibit peroxidase-like activity.
Nanomaterials with peroxidase-like activity have a wide range of practical
applications [14, 48]. For example, as described, it can be used in a colorimetric
reaction for detection of glucose. Another example is that peroxidase enzymes
 Enzyme-Like Activity of Nanomaterials 189
catalytically oxidize biological substrates into products that are less toxic than
the substrate and, therefore, can be utilized for waste water treatment [31].

Oxidases
Oxidase enzymes catalyze oxidation-reduction reactions. This reaction in-
volves reducing molecular oxygen (O2 ), which serves as an electron acceptor,
to oxidize the substrate. The molecular oxygen itself is reduced to water or hy-
drogen peroxide (H2 O2 ). Similar to the peroxidation reaction, the substrate is
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transformed to an oxidized product.

oxidase
O2 + AH −−−−−
−→ H2 O + A
oxidase
O2 + AH + H2 O −−−−−
−→ H2 O2 + A

In most cases, a specific name is given to the oxidase that catalyzes the
oxidation of biological substrates. For example, glucose oxidase, cholesterol ox-
idase, cytochrome c oxidase, cytochrome P450 oxidase, NADPH oxidase, and
xanthine oxidase are the specific oxidase enzymes that catalyze the oxidation
of glucose, cholesterol, cytochrome c, cytochrome P450, NADPH, and xanthine,
respectively.

Superoxide Dismutases
Superoxide is one of the ROS continuously generated in vivo. SOD, com-
monly cofactored with Cu/Zn, Fe, Mn, or Ni metals, catalytically dismutate
superoxide into molecular oxygen. Superoxide dismutases play an important
physiological antioxidant role.
SOD
O·− +
2 + 2H −−−−−
−→ H2 O2 + O2

Catalase
Catalase catalyzes the decomposition of hydrogen peroxide into molecular
oxygen.
catalase
H2 O2 −−−−−
−→ 2 H2 O + O2

DETECTION OF ENZYME-LIKE ACTIVITY OF NANOPARTICLES

To detect the peroxidase-like activity of nanoparticles in a reaction, generally
both the consumption of hydrogen peroxide and the formation of the oxidized
substrate are examined. For examining oxidase-like activity, one frequently
 190 W. He et al.

investigates the decrease of molecular oxygen levels and the formation of the
oxidized substrate. To determine the involvement of catalase-like reaction,
the consumption of hydrogen peroxide and the formation of molecular oxygen
are commonly assessed. For SOD-like catalyzed reactions, the consumption of
superoxide anion radicals and the formation of hydrogen peroxide are deter-
mined.
The commonly used methods for measurement of substrates and prod-
ucts of enzyme-like activities include colorimetric and fluorescence measure-
ments and ESR measurements [16]. For peroxidase and oxidase catalyzed re-
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actions, the colorimetric method is generally used to detect the oxidized prod-
ucts, which are chromophores. The most commonly used peroxidase and oxi-
dase substrates are 3,3,5,5-tetramethylbenzidine (TMB), o-phenylenediamine
(OPD), ABTS, and 3.3’-diaminobenzidine (DAB). Upon peroxidation, TMB is
oxidized into oxTMB, producing a blue color in the solution. Similarly, OPD is
oxidized into the oxidized-OPD, which has a yellow color.
The generation of molecular oxygen as a product can be examined us-
ing a dissolved oxygen meter. The formation of bubbles, molecular oxygen,
indicates a positive result. This easy assay can be seen with the naked eye,
without the aid of instruments. This is because catalase has a very high spe-
cific activity, which produces a visually detectable response. For example, the
catalase-like activity of nano-Co3 O4 was confirmed by using a dissolved oxy-
gen meter to measure the generation of molecular oxygen while H2 O2 was
consumed [13].
To study the enzyme-like activity of nanoparticles, the ESR method
represents a critically important and convenient analytical approach for
zero-time detection of the reactive substrates and products [10, 11, 22–29].
The use of ESR techniques for the study of each type of enzyme-like
activity of nanoparticles is introduced and illustrated in the following
sections.

ENZYME-LIKE ACTIVITY OF CARBON NANOPARTICLES

AND BIOLOGICAL APPLICATIONS
In 2004, Ali and colleagues [49] first demonstrated that a carbon-derived
nanoparticle, fullerene C60 tris-malonic acid, exhibited SOD-like activity. Sim-
ilar to natural SOD enzymes, the reaction catalyzed by these nanoparticles was
through catalytic dismutation of superoxide, regenerating both molecular oxy-
gen and hydrogen peroxide [49]. Ali and colleagues [50] further demonstrated
that six carboxyfullerenes with different electronic and biophysical character-
istics all exhibited SOD activity. In addition, they observed good correlation be-
tween the neuroprotection exerted by carboxyfullerenes and the SOD activity
 Enzyme-Like Activity of Nanomaterials 191
[50]. Quick and associates [51] reported that carboxyfullerene nanoparticles,
C3(e,e,e-C60 (C(COOH2 ))3 ), were SOD mimetics, capable of improving cognition
and extending the lifespan of nontransgenic C57BL/6 mice.
Besides SOD-like activity, carbon nanoparticles also possess peroxidase-
like activity. It was determined that in the presence of H2 O2 , single-walled
carbon nanotubes (SWNTs) showed intrinsic peroxidase-like activity, catalyt-
ically oxidizing the peroxidase substrate TMB into oxTMB, producing a blue
color change [30].
Similar to the commonly used horseradish peroxidase, the catalytic
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peroxidase-like activity of SWNTs is dependent on pH, temperature, and

H2 O2 concentration. Guo and colleagues [33] determined that hemin-graphene
hybrid nanosheets also possess intrinsic peroxidase-like activity. With the
peroxidase-like activity, both SWNTs and hemingraphene hybrid nanosheets
have been used for label-free colorimetric detection of disease-associated
single-nucleotide polymorphisms [30, 33].
Helical carbon nanotubes exhibited high peroxidase-like activity as well
[32]. Because of their strong affinity for H2 O2 , the helical carbon nanotubes
have been utilized as a biocatalyst and amperometric sensor for the detection of
H2 O2 [32]. Liu and colleagues [12] placed Au-nanoparticles on graphene sheets
and found that these new graphene-based nanomaterials exhibit switchable
peroxidase-like activity in response to specific DNA sequences.
Song and associates [36] determined that carboxyl-modified graphene ox-
ide exhibited intrinsic peroxidase-like activity by the observation that in the
presence of hydrogen peroxide, it catalytically oxidized the most commonly
used peroxidase substrate TMB into the blue color oxTMB product. Song and
coauthors [30] developed a simple, cheap, and highly sensitive and selective
colorimetric method for glucose detection in buffer solution or diluted blood
and fruit juice samples.
Kinetic studies indicate that carboxyl-modified graphene oxide has even
higher catalytic activity for TMB than the natural enzyme, horseradish perox-
idase. Further studies indicated that the observed peroxidase-like activity is
not related to the trace amount of metal catalyst in the sample but instead is
an intrinsic property of the modified graphene oxide.
Carbon nanotubes are potentially useful for a variety of applications, in-
cluding for biocatalysis, immunoassay, environmental monitoring, pathogenic
diagnosis, and genetic diseases [32, 33]. A good example is that because of the
peroxidase-like catalytic activity, graphene oxide has been applied for detec-
tion of glucose [36].
Carbon nanodots exhibit intrinsic peroxidase-like activity and can be uti-
lized for glucose detection [52]. At the present, many nanoparticles have been
developed to be used for glucose detection [12, 13, 40, 41, 43, 53–58].
 192 W. He et al.

ENZYME-LIKE ACTIVITY OF NOBLE METAL NANOPARTICLES

Platinum Nanoparticles as Multiple Enzyme Mimics

Among noble metal nanoparticles, Pt has been studied most extensively. Pt
nanoparticles exhibit SOD-like, catalase-like, peroxidase-like, and oxidase-like
activities. As to the oxidase-like activity, Pt nanoparticles have been shown to
act as ascorbic acid oxidase and polyphenol oxidase.
Kajita and coworkers [59] prepared Pt nanoparticles smaller than 5 nm
and stabilized by pectin. The resulting pectin-Pt nanoparticles exhibited both
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SOD-like and catalase-like activity, capable of decomposing H2 O2 and con-

comitantly generating O2 like catalase, and quenching O2 −• like SOD [59]. In
their later studies, the SOD-like and catalase-like activities of Pt nanopar-
ticles were employed to reduce oxidative stress caused by ROS. This led to
an extension of the lifespan of C. elegans [60]. When pretreating human lym-
phoma U937 and HH cells with Pt nanoparticles, the double enzyme activ-
ity of Pt nanoparticles inhibited heat-induced apoptosis in a dose-dependent
manner [61].
Using protein apoferritin as a template, 1–2 nm Pt nanoparticles within
apo-ferritin (Ft-Pt) were synthesized [23, 62]. The Ft-Pt nanoparticles were
very stable and exhibited not only catalase-like activity to catalyze the decom-
position of H2 O2 but also peroxidase-like activity (Figure 1) [23, 62].
Ten nanometer (10 nm) monodispersed Pt nanocubes, prepared by a reduc-
tion method in cetyltrimethylammonium bromide, exhibited peroxidase-like
activity [44]. Pt nanotubes with high porosity were constructed by using tel-
lurium nanowires as a sacrificing template in aqueous solution at room tem-
perature. These Pt nanotubes exhibited intrinsic peroxidase-like activity in the
presence of H2 O2 [63].

Gold Nanoparticles as Multiple Enzyme Mimics

The first report of the enzyme-like activity of Au nanoparticles was in 2004.
Investigators observed that naked 3.6 nm Au nanoparticles exhibited glucose
oxidase-like activity, capable of catalyzing the aerobic oxidation of glucose to
gluconate under mild conditions [64]. Under similar conditions, Cu, Ag, Pd,
and Pt nanoparticles with similar dimension (3–5 nm) did not exhibit glucose
oxidase-like activity. These results inspired follow-up studies on the oxidase-
like activity of Au nanoparticles [56, 65].
Au nanoparticles were also found to possess SOD-like activity, which was
dependent not only on the particle size but also the surface coating [25]. The
tannic acid coated Au nanoparticles exhibited extremely high SOD-like activ-
ity in eliminating superoxide anion radicals. The peroxidase-like activity of
Au nanoparticles was found using positively charged gold nanoparticles, and
 Enzyme-Like Activity of Nanomaterials 193
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Figure 1: Synthesis of Pt-Ft and measurement of the consumption of H2 O2 in the reactions

catalyzed by (i) catalase and (ii) Pt-Ft nanoparticles via hydroxyl radical formation in
H2 O2 /UV system [23].
C Elsevier. Reproduced by permission of Elsevier. Permission to reuse must be obtained from
the rightsholder.

was employed to fabricate a colorimetric method for detection of H2 O2 and glu-

cose [43]. In another study, the peroxidase-like activity of unmodified, amino-
modified, and citrate capped Au nanoparticles was compared, and the results
indicated that unmodified Au nanoparticles exhibited the highest catalytic ac-
tivity [66]. The peroxidase-like activity of Au nanoparticles on the color produc-
tion was affected by additives such as metal ions and antioxidant molecules.
The color intensity was dependent on the dose of the additives, thus a colori-
metric method can be developed for bio-sensing.
The specific enhancing effect of Hg2+ on the peroxidase activity of Au
nanoparticles was used to detect Hg2+ with a detection limit of 3.0 × 10−10
M [67]. A colorimetric assay for dopamine was also implemented for visual
detection due to the visually obvious inhibition of dopamine on the peroxidase-
like activity of bovine serum albumin (BSA) stabilized Au nanoparticles, with
a limit of detection of 10 nM [68]. As a peroxidase mimic, Au nanoparticles
catalyzed chromogenic reactions can also be used to detect H2 O2 or other
molecules whose changes are related to reactions involving H2 O2 . BSA-coated
Au nanoparticles as peroxidase mimetics have been used for sensitive and
 194 W. He et al.

selective detection of H2 O2 , xanthine and uric acid, which react with xanthine
oxidase or uricase, respectively, to produce H2 O2 followed by further detection
[69, 70].

Au@Pt Nanoparticles as Multiple Enzyme Mimics

He and colleagues [24] utilized Au nanorods as a template to modulate the
growth of Pt nanodots on Au nanorods, forming Au@Pt core/shell nanostruc-
tures, which endowed the whole structure with good optical response and a
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well-dispersed surface distribution of Pt nanodots. In one study, they verified

that Au@Pt nanostructures exhibited multiple enzyme mimetic capability, for
example, oxidase, peroxidase, and catalase mimetic activity. The oxidase-like
activity was evaluated using the typical substrates TMB and OPD in the ab-
sence and presence of molecular oxygen. Similarly, the peroxidase-like activity
was evaluated with TMB and OPD in the absence and presence of H2 O2. In
both cases, in presence of Au@Pt and H2 O2 , TMB and OPD were oxidized to
blue color and yellow color oxidized products, respectively (Figure 2C and 2D).
The chemical mechanisms of the formation of oxidized-TMB by oxidation with
oxygen and with H2 O2 are shown in Figure 2E and 2F [24].
Au@Pt nanorods (NRs) with oxidase-like and peroxidase-like activities
have been applied in immunoassays. As shown in Figure 3, for the Au@Pt
NRs-based enzyme linked immunosorbent assay (ELISA), antigen was first
bound to the capture antibody and then detected by the Au@Pt NRs-conjugated
detection-antibody via the oxidation of TMB. The Au@Pt-based ELISA is sen-
sitive to the concentration of mouse IL-2 antigen both in the presence and
absence of hydrogen peroxide (Figure 3).
The catalase-like activity of Au@Pt nanoparticles was first determined by
measuring the decrease of the reactant, H2 O2 , using ESR techniques [26]. As
previously reported, the quantity of H2 O2 in the reaction was determined us-
ing an H2 O2 /UV system to generate hydroxyl radicals, which were trapped
by the 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO) spin trap.
Due to the consumption of H2 O2 , a smaller quantity of hydroxyl radicals was
generated and thus resulted in weaker ESR signal intensities. The decrease in
signal intensity is also dependent on the Au@Pt concentration (Figure 4B). As
a comparison, similar results were obtained when a catalase was tested in the
same system.
The catalase-like activity of Au@Pt nanoparticles was further confirmed
using the ESR spectroscopy oximetry technique. By employing the ESR oxime-
try method [24], the catalase-like activity of Au@Pt nanoparticles was mea-
sured by the decomposition of hydrogen peroxide to produce molecular oxygen.
As shown in Figure 5B, Au@Pt can catalyze the decomposition of hydrogen
peroxide to produce the formation of molecular oxygen. The results clearly in-
dicate that oxygen generation is dependent on the H2 O2 concentration (Figure
 Enzyme-Like Activity of Nanomaterials 195
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Figure 2: SEM image (a) and UV-vis-NIR absorption spectra (b) of Au@Pt nanostructures
(inset in b is a schematic picture of the nanostructure with dotted lines representing Pt
nanodots). The absorption spectrum of the inner Au NRs (black line) is shown for comparison.
Color evolution of TMB and OPD oxidation in the absence (c) and presence (d) of H2 O2 in a
pH 4.5 PBS buffer. Corresponding reaction mechanisms for O2 (e) and H2 O2 (f) reduction
with TMB [24].
C Elsevier. Reproduced by permission of Elsevier. Permission to reuse must be obtained from
the rightsholder.

5B) [24]. In addition, oxygen bubbles were visibly observed in the capillary
tubes with samples containing catalase or Au@Pt nanoparticles (Figure 5C).
More bubbles continued to be generated long after termination of the ESR
experiment.
 196 W. He et al.
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Figure 3: (a) Au@Pt NRs-based ELISA. Antigen is first bound to capture antibody and then
detected by the Au@Pt NR-conjugated detection antibody via the oxidation of TMB. Both
the oxidase- and peroxidase-like activities of the NRs can be employed103 (b). Au@Pt
0.17-based ELISA is sensitive to the concentration of mouse IL-2 antigen both in the presence
and absence of hydrogen peroxide [24].
C Elsevier. Reproduced by permission of Elsevier. Permission to reuse must be obtained from
the rightsholder.

The mechanism underlying the enzyme-like activity of Au@Pt nanopar-

ticles involves the ability of Pt nanoparticles to effectively catalyze electron
transfer and the reduction of oxygen, H2 O2 reduction, and decomposition. This
study also showed that Au@Pt nanoparticles have a highly stable catalytic
 Enzyme-Like Activity of Nanomaterials 197
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Figure 4: The effect of (a) catalase and (b) Au@Pt nanostructures on the formation of
hydroxyl radical in H2 O2 /UV system. Samples were mixture of 20 mM BMPO, 5 mM H2 O2 , and
various concentrations of catalase or Au@Pt in PBS and then exposed to 15 min UV light at
270 nm. Addition of catalase/Au@Pt reduced the ESR signal intensity in a
concentration-dependent manner [24].
C Elsevier. Reproduced by permission of Elsevier. Permission to reuse must be obtained from
the rightsholder.

activity even under extreme conditions of pH and temperature. Using Au@Pt

nanoparticles in place of horseradish peroxidase, an Au@Pt nanoparticle-based
ELISA was established and used for the detection of mouse interleukin 2,
which can work even without H2 O2 due to the oxidase-like activity of Au@Pt.
A similar sandwich ELISA based on irregular-shaped Pt nanoparticles was
developed in another study for detection of rabbit IgG [71].
Such Au@Pt nanorods were further demonstrated to be kinetically similar
to ascorbic acid oxidase during oxidation of ascorbic acid. Through ESR analy-
sis, Zhou and colleagues [29] found that Au@Pt nanorods did not scavenge hy-
droxyl radicals but diminished the antioxidant ability of ascorbic acid for scav-
enging hydroxyl radicals. Moreover, the Au@Pt nanorods reduced the ability of
ascorbic acid to scavenge 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radicals and
superoxide radicals [29]. This effect on ascorbic acid’s antioxidant activity was
shown to result from decomposition of ascorbic acid catalyzed by Au@Pt, an ac-
tivity analogous with ascorbic acid oxidase. Liu and associates [72] developed
a method to screen the inhibitor for the oxidase mimics of Au@Pt nanorods.
They found that Fe2+, Cu2+, Hg2+, and NaN3 exhibited an inhibitory effect;
Fe2+ ions thus cause irreversible inhibition while Cu2+, Hg2+, and NaN3 cause
reversible inhibition. The inhibition effect is dependent on the concentration
 198 W. He et al.
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Figure 5: The catalase-like activity of Au@Pt nanostructures. Oxygen generation is measured

by ESR oximetry experiments in a closed chamber with samples of 0.1 mM 15N-PDT, 20
unit/ml catalase (A) or 1 nM Au@Pt (B) mixed with different concentration of (black) without;
(red) 0.125 mM; (green) 0.25 mM; and (blue) 0.5 mM of H2 O2 in PBS. (C) Observation of the
dioxygen bubbles generated in H2 O2 decomposition without catalyst (a) and catalyzed by
catalase (b) or Au@Pt nanostructures (c). (For interpretation of the references to color in this
figure legend, the reader is referred to the Web version of this article) [24].
C Elsevier. Reproduced by permission of Elsevier. Permission to reuse must be obtained from
the rightsholder.

of metal ions, a method for detection of Hg2+ with LOD 5.5 × 10−7 M was es-
tablished. By employing the peroxidase-like activity of Au@Pt nanoparticles, it
is possible to construct a platform for detection of bioactive molecules involv-
ing generation or consumption of H2 O2 , such as H2 O2 , ascorbic acid, glucose,
cholesterol and acetylcholine, etc. Detection of Escherichia coli with a limit of
detection of 7 cfu/mL was achieved using a TMB colorimetric method based
on the peroxidase-like activity of 4-mercaptophenylboronic acid coated Au@Pt
nanoparticles [73].
Similar to the most commonly used natural horseradish peroxidase, a
nanoparticle with a peroxidase-like enzyme activity and a peroxidase sub-
strate, for example, a chromogenic donor, can be used for detection of hy-
drogen peroxide. In the presence of glucose oxidase, the nanoparticles with
 Enzyme-Like Activity of Nanomaterials 199
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Figure 6: AgAu, AgPd, and AgPt nanoparticles (NPs) with different shapes. Lower left panel
shows the color evolution for oxidation of OPD, TMB, and ABTS by Pd or AgPd nanoparticles.
Panel b shows the dependence of OPD oxidation on the Ag/Pd ratio. The purple line
represents the value of the control experiment [45].
C American Chemical Society Reproduced by permission of American Chemical Society.
Permission to reuse must be obtained from the rightsholder.

peroxidase-like activity can also be used for detection of glucose. In addition,

peroxidase-like nanoparticles conjugated to immunoglobulins can be used as
immunohistological probes for the detection of tissue antigens. Furthermore,
in an enzyme amplified immunoassay system, it can be used for quantitation
of antigens [24].

Other Bimetallic Alloy Nanoparticles as Enzyme Mimetics

He and colleagues [45] synthesized a series of bimetallic nanoparticles
with alloyed structures and demonstrated that these nanoparticles possess
peroxidase-like and oxidase-like activity. They further demonstrated that the
enzyme-like activity of bimetallic alloy nanoparticles is determined by the al-
loy compositions. This finding provided an effective way to tune the enzyme
mimetic activity by changing the alloy compositions. They synthesized three
AgM (M = Au, Pd, Pt) bimetallic alloy nanostructures and determined that
these three Ag based bimetallic nanostructures exhibited similar peroxidase-
like activity, capable of oxidizing TMB into the blue color oxTMB product in the
presence of H2 O2 [45]. Using AgPd nanoparticles with different Ag/Pd ratio,
they observed the peroxidase-like activity of AgPd nanoparticles, and the en-
zyme activity was highly dependent on the composition of the alloy (Figure 6).
Furthermore, Hu and colleagues [26] synthesized four Au@PtAg core/shell
nanorods with different Ag percentages. They found that these four Au@PtAg
nanorods all exhibited peroxidase-like, oxidase-like, catalase-like, and SOD-
like activities, and that their enzyme activities decreased proportionally with
an increased Ag percentage in the alloy (Figure 7). By employing the ESR
 200 W. He et al.
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Figure 7: Ag/Pt molar ratio in different Au@PtAg NPs measured by EDX (A) and highly Ag/Pt
ratio dependence on Au@PtAg NRs’ peroxidase (B), catalase (C), and SOD (D) like activity
[26].
C Royal Society of Chemistry. Reproduced by permission of Royal Society of Chemistry.
Permission to reuse must be obtained from the rightsholder.

oximetry method, Hu and colleagues [26] determined the catalase-like activity

of Au@PtAg alloy nanoparticles of different Ag/Pt ratios (0.33, 0.5, and 0.67)
by measuring the decomposition of hydrogen peroxide to produce molecular
oxygen. As shown in Figure 7C, Au@PtAg can catalyze the decomposition of
hydrogen peroxide to produce molecular oxygen. The results indicate that oxy-
gen generation is dependent on the Au@PtAg alloy nanoparticle composition:
a higher Ag/Pt ratio leads to lower catalase-like activity (Figure 7C).
In the case of Au@PtPd nanoparticles, tuning oxidase-like activity through
changing Pt/Pd composition alloy was also achieved [28]. With equivalent com-
position, the shape would work as an important factor. Dong and coworkers
[74] reported that PdPt nanosponges possessed much better oxidase and per-
oxidase mimetics than those of PdPt nanowires.
In addition to measurement of the formation of molecular oxygen to deter-
mine the catalase-like activity, the catalase-like activity of the Au@PtAg alloy
 Enzyme-Like Activity of Nanomaterials 201
nanoparticles was further confirmed by measuring the decrease of the reactant
H2 O2 using ESR techniques [26]. As previously reported, the quantity of H2 O2
in the reaction was determined using an H2 O2 /UV system to generate hydroxyl
radicals, which were trapped by the BMPO spin trap. Due to the consumption
of H2 O2 , a lower quantity of hydroxyl radicals was generated and thus resulted
in weaker ESR signal intensities.
Bimetallic nanoparticles in a cage of apo-ferritein (Aft-FePt) have been
synthesized and were found to exhibit peroxidase-like activity. It was demon-
strated that AftFePt were more active than monometallic counterparts Aft-
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Fe and Aft-Pt because of the confinement of Pt over Fe domains [62]. Apart

from noble metal nanoparticles, non-noble metal-based bimetallic alloy FeCo
nanoparticles have been prepared by a simple co-reduction method [22]. The
FeCo nanoparticles showed both oxidase and peroxidase activity, and were
more active than Fe and Co monometallic nanoparticles due to synergis-
tic effects between the two metals. Hydroxyl radical generation in the re-
action between CoFe nanoparticles and H2 O2 was verified by ESR. Gener-
ation of hydroxyl radicals appeared to be implicated in the peroxidase like
activity [22].

IRON AND COPPER NANOPARTICLES AS ENZYME MIMETICS

Gao and colleagues [31] were the first to determine that Fe3 O4 mag-
netic nanoparticles possess an intrinsic peroxidase-like activity. Similar to
horseradish peroxidase, Fe3 O4 magnetic nanoparticles catalyzed the oxida-
tion of three peroxidase substrates, TMB, DAB, and OPD. Fe3 O4 magnetic
nanoparticles had a lower Michaelis constant for TMB than horseradish per-
oxidase. Horseradish peroxidase has a higher affinity to the substrate H2 O2 .
Tested with TMB, Fe3 O4 nanoparticles showed a higher catalytic activity than
horseradish peroxidase. Similar to horseradish peroxidase, the catalytic activ-
ity of Fe3 O4 nanoparticles is dependent on pH, temperature, and H2 O2 concen-
tration [31]. In addition to utilizing nano-Fe3 O4 as a novel immunoassay, Gao
and colleagues [31] proposed that nano-Fe3 O4 can be utilized for many applica-
tions for medicine, diagnostics, biotechnology, and in bioremediation of waste
water.
Wei and associates [40] demonstrated that in the presence of H2 O2, nano-
Fe3 O4 catalyzed the oxidation of the peroxidase substrate, 2,2 -azino-bis(3-
ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and also sug-
gested that nano-Fe3 O4 can be used in a colorimetric method for detection of
H2 O2 . In addition, Wei and associates [40] also determined that nano-Fe3 O4
can serve as a sensitive and selective method for glucose detection.
Using TMB as the peroxidase substrate and in the presence of H2 O2 , Liu
and coauthors [38] determined that all the three nano-Fe3 O4 nanocrystals
(cluster spheres, octahedra, and triangular plates) exhibited peroxidase-like
 202 W. He et al.

activity. The potency of the peroxidase-like activity followed the order: clus-
ter spheres > triangular plates > octahedral. These results indicated that the
enzyme activity was dependent on the structure of nano-Fe3 O4.
Iron oxide nanoparticles coated with six different coating agents were syn-
thesized and their peroxidase-like activity was evaluated using glucose detec-
tion as a model system for determination [41]. The findings suggest that coat-
ing iron oxide nanoparticles can improve peroxidase-like activity.
A magnetoferritin, consisting of a protein shell composed of apoferritin sur-
rounded with magnetic Fe3 O4 or γ-Fe2 O3 nanoparticles, has been synthesized.
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Magnetoferritin was determined to possess very high peroxidase-like activity

[39].
A number of iron-derived nanoparticles have been determined to pos-
sess peroxidase-like activity. These include manganese ferrite (MnFe2 O4 )
nanoparticles [37], Prussian blue modified g-Fe2 O3 magnetic nanoparticles
[42], CoFe2 O4 nanoparticles [52], ZnFe2 O4 nanoparticles [58], FeS nanopar-
ticles [75], FeSe nanoparticles [75], and two iron(III)-based metal–organic
nanoparticles [46]. Thus, those nanoparticles that possess peroxidase-like ac-
tivity can also be utilized for glucose detection. For example, ZnFe2 O4 magnetic
nanoparticles were used to detect glucose in urine [58].
In the presence of H2 O2 , cupric oxide nanoparticles (nano-CuO) catalyze
the oxidation of TMB into the blue colored product oxTMB, indicating that
nano-CuO exhibits peroxidase-like activity and can be used for detection of
H2 O2 and glucose [15, 76]. He and colleagues [77] found that CuS concave
polyhedral superstructures exhibited intrinsic peroxidase-like activity, catalyt-
ically oxidizing TMB and OPD in the presence of hydrogen peroxide. Dutta and
associates [53] reported that CuS nanoparticles possess intrinsic peroxidase-
like activity and can be utilized to monitor glucose levels in human blood glu-
cose. Cu9 S5 nanocrystals coated on polyaniline nanowires were also found to
exhibit peroxidase-like activity [78].
Cu nanoclusters are also found to possess intrinsic peroxidase-like activity
[77]. Similar to naturally occurring peroxidase, Cu nanoclusters can catalyze
the oxidation of TMB by H2 O2 . A colorimetric method for glucose detection was
also developed [26].

OTHER METAL NANOPARTICLES AS ENZYME MIMETICS

Ceria nanoparticles exhibit oxidase-like activity at acidic pH that can be used
in immunoassays (ELISA) [47, 79]. Cerium oxide nanoparticles (nanoceria)
have been shown to exhibit SOD activity as evidenced by EPR analysis [80].
It was determined that the surface oxidation state of nanoceria and cerium
(III) concentrations at the surface of the particles play an important role in the
SOD-like activity [80].
 Enzyme-Like Activity of Nanomaterials 203
Co3 O4 nanoparticles (nano-Co3 O4 ) catalyzed the oxidation of the peroxi-
dase substrate TMB by H2 O2 to produce the blue colored OxTMB [13]. The
peroxidation reaction catalyzed by nano-Co3 O4 is similar to the standardized
peroxidation reaction using the natural horseradish peroxide, with the cat-
alytic activity dependent on pH, temperature, and H2 O2 concentration [81].
It was determined that at high H2 O2 concentration, the catalytic activity of
horseradish peroxide is inhibited. However, at high H2 O2 concentration, the
catalytic activity of nano-Co3 O4 is more stable than that of horseradish perox-
ide.
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Based on the results observed, Mu and colleagues [13] suggested that

nano-Co3 O4 exhibits electrocatalytic activity. It facilitates the electron trans-
fer of the peroxidase from the substrate TMB and H2 O2 as follows. Once the
TMB is absorbed on the surface of nano-Co3 O4 , the lone-pair electrons from
the amino group of TMB are transferred to the nano-Co3 O4 moiety resulting in
both increase electron density and mobility in the TMB-absorbed nano-Co3 O4 .
As such, this electronic transformation in the TMB-absorbed nano-Co3 O4 facil-
itates the electron donated to H2 O2 . The overall catalytic effect of nano-Co3 O4
is to help transfer electron(s) from the peroxidase substrate (s), including TMB,
to H2 O2 [13]. The catalase-like activity of nano-Co3 O4 was confirmed by using
a dissolved oxygen meter to measure the generation of molecular oxygen dur-
ing the consumption of H2 O2 catalyzed by nano-Co3 O4 [13]. Mu and colleagues
[13] demonstrated that nano-Co3 O4 can be employed for colorimetric determi-
nation of H2 O2 and glucose.
Besides peroxidase-like and catalase-like activities, Dong and associates
[82] reported that nano-Co3 O4 also possessed intrinsic SOD-like activity, with
the potency of all these three enzyme-like activities much higher than nano-
Fe3 O4 [82]. Different from nano-Fe3 O4 , nano-Co3 O4 did not follow a clas-
sical Fenton reaction with hydrogen peroxide. This mechanistic difference
was attributed to the high redox potential of Co3+/Co2+. Based on the high
peroxidase-like activity, a new immunohistochemical assay was developed in
which an avastin antibody was conjugated onto the surface of nano-Co3 O4 [82].
Ag nanoparticles stabilized by chitosan were found to exhibit peroxidase-
like activity [2]. A silver-based colorimetric method for detection of glucose was
designed with a detection limit of 100 nM. Au nanoparticles coated with ele-
mental Bi, Pd, and Ag in the form of a nanoshell have been determined to have
highly enhanced peroxidase-like activity compared with the monometallic Au
counterparts. This enhancement of peroxidase-like activity comes from the
shell metal and was used for fluorescent detection of thrombin, the herbicide
bensulfuron-methyl, and acetylcholine [83–85]. Compared with Au nanoparti-
cles and Pt nanoparticles, Ag, Cu, and Bi nanoparticles are easily dissolved
and oxidized to the corresponding metal ions by molecular oxygen and hy-
drogen peroxide. This ease of dissociation into metal ions may explain their
enzyme-like activities.
 204 W. He et al.

Many other nanoparticles have been reported to exhibit peroxidase-

like activity. These include nano-V2 O5 [86], Au(core)Pd(shell) (Au@Pd)
bimetallic nanoparticles dispersed on grapheme [87], three tetranu-
clear zirconium-substituted nanomaterials [88], tungsten carbide nanorods
[89], Fe3 O4 -Au nanomaterials [90], platinum modified Fe3 O4 magnetic
nanoparticles (Fe3 O4 @Pt NPs) [91], CdS nanoparticles [92], porous Co3 O4
nanorods–reduced graphene oxide [93], TiO2 nanotube arrays [94], multi-
walled carbon nanotubes and nanoparticles [95], and VO2 (B) nanobelts
[57]. For applications, VO2 (B) nanobelts and TiO2 nanotube arrays can
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be used in a colorimetric assay to detect glucose and hydrogen peroxide

[57, 94].

PERSPECTIVES
As addressed in this review, a variety of nanoparticles exhibit enzyme-like ac-
tivities, including peroxidase-like, oxidase-like, catalase-like, and superoxide
dismutase-like activities. Because of these activities, the nanoparticles with
enzyme-like activities, with the peroxidase-like activity in particular, have
high potential for being developed for many applications. These include for
medical diagnosis, such as detection of glucose and hydrogen peroxide, im-
munoassays, removal of environmental pollutants, and in waste water treat-
ment. This is relatively a new field. It is anticipated that many more new
nanoparticles with highly efficient enzyme-like activities will be synthesized
to expand their applications.
While there has been rapid progress in developing nanomaterials with en-
zyme mimetic activity, it remains unclear how widely applicable these ma-
terials will be for in vivo and clinical purposes. To date, it is not certain
whether nanoparticles with enzyme-like activity can significantly replace uses
of many naturally occurring enzymes. Additional efforts are needed to address
two important issues: efficacy and safety. All the enzymes in our body work
interdependently as a holistic system. Any artificial alternatives, including
those from nanoparticles, may not well fit to the system and may cause se-
vere side effects. In addition, overproduction of ROS can be harmful to our
health, through inducing oxidative stress, leading to lipid peroxidation, and
causing DNA damage [5, 96–100]. During the expression of enzyme-like func-
tions, the nanoparticles can generate free radicals, causing toxicological effects.
Chen and colleagues [16] have demonstrated that while iron oxide nanoparti-
cles exert peroxidase-like and catalase-like activities, hydroxyl radicals were
generated and lead to concentration-dependent cytotoxicity on human glioma
U251 cells. Thus, full utilization of the beneficial effects of enzyme-like ac-
tivity of nanoparticles warrants further research to explore the usefulness in
vivo.
 Enzyme-Like Activity of Nanomaterials 205

ACKNOWLEDGEMENT
We thank Dr. Frederick A. Beland for critical review of this manuscript. This
article is not an official U.S. Food and Drug Administration (FDA) guidance
or policy statement. No official support or endorsement by the U.S. FDA is
intended or should be inferred.

FUNDING
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W. He thanks the National Natural Science Foundation of China (Grant No.

201303153) for support.

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