Nano Research

https://doi.org/10.1007/s12274-018-2079-8

Prussian blue with intrinsic heme-like structure as

peroxidase mimic
Jinxing Chen1,2, Qingqing Wang1, Liang Huang1,2, Hui Zhang1,3, Kai Rong1,2, He Zhang1,2, and Shaojun
Dong1,2,3 ()

1
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences,
Changchun 130022, China
2
University of Science and Technology of China, Hefei 230026, China
3
University of Chinese Academy of Sciences, Beijing 100049, China

Received: 17 March 2018 ABSTRACT

Revised: 20 April 2018
Accepted: 22 April 2018
© Tsinghua University Press
and Springer-Verlag GmbH
Germany, part of Springer
Nature 2018
KEYWORDS
Prussian blue,
Prussian blue analogs,
peroxidase,
high-valent iron-oxo
Despite the intrinsic peroxidase-like activity of Prussian blue nanoparticles (PBNPs),
their enzyme-mimic mechanism has been scarcely investigated to date. Herein,
we probed the catalytic site of PBNPs for the first time, by comparing their
peroxidase-like activity with that of a series of Prussian blue analogs (PBAs) in
which Fe atoms were replaced by Co, Ni, and Cu. The PBNPs exhibited the
highest maximal reaction velocity (1.941 μM·s−1), which was at least 13 times
higher than that of the PBAs, demonstrating that the peroxidase-like properties of
PBNPs could be ascribed to the FeNx (x = 4–6) instead of the FeC6 units. Notably,
the PBNPs/H2O2 couple also showed much higher oxidizability than ·OH radicals
produced from the Fenton reaction, implying that a high active Fe(IV)=O
intermediate might be formed in the FeNx units. This study can thus pave the
way for the wider application of PBNPs in biomimetic reactions.

1 Introduction been synthesized to mimic the structure and functions

Heme enzymes such as horseradish peroxidase (HRP)
and cytochrome P450 are powerful biocatalysts in natural
systems [1, 2], capable of effectively hydroxylating
strong C−H or C=C bonds, and high-valent iron-oxo
intermediates (e.g., Fe(IV)=O or Fe(IV)−OH) were
identified as reactive species in the catalytic cycles [3,
4]. Inspired by the high activity of heme, a large number
of ferric tetraamido macrocyclic ligand (TAML)- and
tetramethylcyclam (TMC) ligand-based complexes have
of heme enzymes [5–8]. Notably, Fe(IV)=O or more active
Fe(V)=O species can also be generated all-pervasively
by the reactions of Fe-TMAL and Fe-TMC complexes with
H2O2 [9] or other oxidants, such as m-chloroperbenzoic
acid (mCPBA) [10], iodosylbenzene (PhIO) [11], and
NaClO [12]. These active species have been widely
applied in olefin cyclopropanation [13, 14], C−H bond
hydroxylation [15–17], and even water oxidation
reactions [18].
Besides the Fe-TAML complexes discussed above,

Address correspondence to dongsj@ciac.ac.cn

2 Nano Res.
nanoparticles (NPs) (especially the Fe3O4 NPs reported
by Yan et al. [19]) were also found to possess intrinsic
peroxidase-like activity. Although many nanomaterials
with peroxidase-like activities have been reported to date,
their catalytic mechanisms have rarely been investigated
in detail [20–25]. Among these nanozymes, Fe-containing
nanoparticles have been studied most extensively,
due to their relatively high peroxidase-like activity
[26–32]. In 2014, Zhang et al. reported that FeIII and
FeII ions have higher peroxidase-like activity than Fe3O4
NPs, even when the size of the latter NPs was reduced
to 6 nm, suggesting that the catalytic activity of Fe3O4
NPs originates from surface Fe atoms, which catalyze
the dissociation of H2O2 into ·OH radical via the Fenton
reaction [33].
Prussian blue nanoparticles (PBNPs), as Fe-containing
pigments, were also found to possess peroxidase-like
activity [34–36]. Recently, Willner et al. reported that
PBNPs could catalyze the oxidation of dopamine or
NADH more effectively, compared to their cyanometalate
structural analogs [36]. Furthermore, we discovered
that the peroxidase-like activity of PBNPs was higher
than that of FeIII. These results revealed that the catalytic
capacity of PBNPs did not simply originate from the
Fenton reaction. In this study, we introduced different
divalent transition metal ions (i.e., CoII, NiII, and CuII)
to replace N atom-coordinated FeIII species. The catalytic
site of the PBNPs has been identified by comparing
their catalytic activities with those of a series of Prussian
blue analogs (PBAs). Inspired by the plane quadrilateral
structure of heme, we propose that the catalytic mechanism
of the PBNPs was also similar to that of heme enzymes.
2 Results and discussion
The formation of PBNPs can be considered as a self-
assembly process (Scheme 1(a)). In K4[Fe(CN)6], FeII
atoms are six-fold coordinated to the C atoms of the
C≡N ligands, and the isolated electron pairs at N atoms
of C≡N ligands are octahedrally combined with the
guest FeIII to form cyanide-bridged cubic networks.
Moreover, unsaturated FeN5 and FeN4 units could
be formed, due to the existence of [FeII(CN)6] vacancies
(Fig. S1(b) in the Electronic Supplementary Material
(ESM) [37]. PBAs were synthesized though the same
methods used for the PBNPs. The FeNx, CoNx, NiNx,
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Scheme 1 (a) Assembly process of PBNPs. (b) Structure of
FeNx unit in PBNPs. (c) Structure of MNx unit in PBAs. The blue
and yellow bands represent the macrocyclic assembly of four
N≡C ligands in the same plane. X and Y represent H2O or N≡C
ligands.
and CuNx units in the corresponding frameworks
have a planar macrocyclic substructure, similar to
that of heme and nonheme Fe-TAML-based complexes
(Scheme 1(c)).
According to the N- and C-coordinated metal atoms
(M1 and M2, respectively, in M1–N≡C–M2), the PBNPs
and PBAs prepared in this work were denoted as FeFe,
CoFe, NiFe, CuFe, FeCo, CoCo, NiCo, and CuCo. Based
on the different metal atoms coordinated with C atoms
in C≡N bridges, the cubic frameworks were classified
into two groups: The first included FeFe, CoFe, NiFe,
and CuFe, all of which contained the FeC6 units,
while the other group consisted of FeCo, CoCo, NiCo,
CuCo, based on the CoC6 units. The particle sizes of
these nanomaterials were controlled by changing the
reaction conditions (Table S1 in the ESM). Transmission
electron microscopy (TEM) images showed that all
nanoparticles were of cubic shape (Fig. 1). The size
distribution of FeCo NPs was centered at 450 nm,
whereas the sizes of the other nanomaterials were in
the 50–120 nm range. Scanning electron microscopy
(SEM) images further showed that these nanocubes
have narrow size distribution and ideal dispersity
(Fig. S2 in the ESM).
The crystal structures of the PBNPs and PBAs were
determined through X-ray diffraction (XRD) measurements.
The XRD patterns of all nanocubes were well consistent
with the standard patterns of face-center-cubic PBNPs
(JCPDS No. 73-0687) [38]. No other phases or impurities
Nano Res. 3

Figure 1 TEM images of FeFe (a), CoFe (b), NiFe (c), CuFe (d), FeCo (e), CoCo (f), NiCo (g), and CuCo (h).

were detected, indicating that pure crystalline phases
of PBNPs and PBAs were synthesized (Fig. 2). The
Fourier-transform infrared (FT-IR) spectra of the PBNPs
and PBAs exhibited a sharp characteristic C≡N stretching
vibration in the 2,000–2,200 cm−1 range, which can be
assigned to the ν(M1–C≡N–M2) mode in the corresponding
nanomaterials (Fig. S3 in the ESM) [39, 40]. The XRD
patterns and FT-IR spectra revealed that the PBNPs
and PBAs had similar framework structures.
X-ray photoelectron spectroscopy (XPS) analysis was
performed to determine the detailed compositions of
the as-prepared catalysts. The Fe, Co, Ni, and Cu 2p
spectra demonstrated the presence of metal components
in the corresponding PBAs (Fig. S4 in the ESM). The
C 1s curves can be deconvoluted into two peaks at
285.4 and 284.5 eV, corresponding to C≡N and CC
groups, respectively (Fig. S5 in the ESM). In order to
investigate the coordination properties of the C≡N
ligands, we compared the binding energies of Fe and
N in PBNPs and hemin. Two peaks at 721.6 eV (Fe 2p1/2)
and 713.1 eV (Fe 2p3/2) in the high-resolution Fe 2p
spectrum were ascribed to the FeNx unit, while the
peak at 708.4 eV (Fe 2p3/2) can be attributed to the
[FeII(CN)6] units of PBNPs (Fig. 3(a)). The binding
energy of Fe was lower in hemin than in PBNPs,
suggesting a slightly higher electron cloud density.
Consisting with the negative shift of the hemin Fe peaks,
the N 1s binding energy showed a positive shift from
397.5 in PBNPs to 398.2 eV in hemin (Fig. 3(b)). The
N 1s spectrum provided direct evidence that all N
atoms of the as-prepared nanomaterials and hemin
were pyridinic (around 398 eV); these atoms are vital
components of N-doped catalysts, due to their specific
coordination ability (Fig. S5 in the ESM) [41–43].
The peroxidase-like activities of PBNPs and PBAs were
determined by colorimetric assays (Fig. 4). In the presence
of H2O2, the as-prepared nanoparticles could catalyze
the oxidation of colorless 3,3’,5,5’-tetramethylbenzidine

Figure 2 XRD patterns of (a) FeFe, CoFe, NiFe, CuFe, and (b) FeCo, CoCo, NiCo, and CuCo.

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4 Nano Res.

Figure 3 (a) Fe 2p and (b) N 1s XPS spectra of PBNPs and hemin.

Figure 4 (a) Photographs of FeFe, CoFe, NiFe, CuFe, FeCo, CoCo, NiCo, and CuCo NPs (from left to right), at 2 mg·mL−1 in water.
(b) Photographs of the oxidation of TMB by the above nanomaterials, along with a blank sample shown at the right end of the sequence.
(c) and (g) Absorption spectra of TMBox. (d) and (h) Time-dependent absorbance changes of TMB at 652 nm. (e) and (i) Steady-state
kinetic assay in the presence of 10 mM H2O2 and varying TMB concentration. (f) and (j) Steady-state kinetic assay in the presence of
1 mM TMB and varying H2O2 concentration. All experiments were conducted in 100 mM HAc-NaAc buffer at pH 4.0 and 20 °C. The
catalyst concentration was 5 μg·mL−1.

(TMB) to the blue oxidized form, TMBox (Figs. 4(b) and
4(c)). Real-time monitoring of the oxidation of TMB was
performed by measuring the absorbance changes at
λ = 652 nm. CoFe, NiFe, and CuFe showed catalytic
activities similar to each other, but also significantly
lower than that of FeFe (Fig. 4(d)). Typical Michaelis–
Menten curves were obtained by varying the concentration
of TMB (or H2O2) while keeping the H2O2 (or TMB)
concentration constant. The initial velocity increased
rapidly with increasing H2O2 or TMB concentration
when FeFe was used as the catalyst (Figs. 4(e) and 4(f)),
whereas its increase was very slow in the case of CoFe,
NiFe, and CuFe. These effects can be understood by
analyzing the structures of the catalysts. Since FeFe,
CoFe, NiFe, and CuFe all had face-center-cubic frameworks,
the only difference between these nanomaterials was
that their N atoms were specifically coordinated with
Fe, Co, Ni, and Cu, respectively. Therefore, the outstanding
catalytic capacity of FeFe mainly originated from the
FeNx unit.

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Nano Res. 5

It is worth noting that CoFe, NiFe, and CuFe also TMB and H2O2 substrates were only slightly different
showed little catalytic activity. However, it is not clear from those of HRP, implying that FeFe and HRP have
whether their catalytic activities derived from FeC6 or similar affinity to TMB and H2O2.
MNx (M = Co, Ni, and Cu). In order to identify their In a previous study, it was suggested that the
catalytic sites, another group of PBAs containing FeCo, peroxidase-like activity of PBNPs can be ascribed to
CoCo, NiCo, and CuCo were studied under the same the high redox potential of Berlin green (BG)/Prussian
experimental conditions. Unsurprisingly, FeCo, based yellow (PY) [35]. However, as the PY/BG transition
on the FeNx unit, exhibited evident superiority than occurred at the FeC6 unit (FeIIIC6/ FeIIC6), this assumption
its counterparts (Figs. 4(g)–4(j)). CoCo and NiCo showed was not consistent with the experimental conclusion
no detectable catalytic activity, suggesting that the that the FeNx unit is the active site. Moreover, no
CoNx, NiNx, and CoC6 units had almost no activity, absorbance change was observed for the PBNPs in
whereas the weak catalytic ability of CuCo originated the absence or presence of H2O2, suggesting that the
from CuNx. In the case of CoFe, NiFe, and CuFe, the oxidation of PBNPs by H2O2 was limited in kinetics
catalytic ability mainly originated from FeC6. To sum (Fig. S11 in the ESM). Based on the high activity of
heme and Fe-TMAL complexes, the peroxidase-like
up, the catalytic properties of these substructures
activity of the PBNPs might be attributed to the
followed the order FeNx >> FeC6 > CuNx. However,
high-valence form of the Fe(IV)=O intermediate.
the NiNx, CoNx and CoC6 units did not provide any
In fact, FeII and FeIII ions can catalyze the decomposition
catalytic activity.
of H2O2 into ·OH radicals and exhibit intrinsic
The maximum initial velocity (Vmax) and Michaelis–
peroxidase-like activity via the Fenton reaction [44]
Menten constant (Km) were obtained from a Lineweaver–
Burk plot derived from the Michaelis–Menten curve Fe2+ + H2O2 → Fe3+ + OH− + ·OH (1)
(Figs. S6–S10 in the ESM) and are listed in Table 1.
The Km parameter generally indicates the affinity Fe3+ + H2O2 → Fe2+ + H+ + ·O2H (2)
between substrates and enzymes [31]. The Km values
Since the Fe(IV)=O species producing route was a more
of the present catalysts showed only little differences,
effective catalytic path than the Fenton reaction, we
because the different ions in their frameworks have a
compared the peroxidase-like activities of PBNPs and
slightly different affinity to TMB and H2O2. However,
FeIII ions (Figs. 5(a) and 5(b)). The FeIII ions exhibited
the Vmax values of FeFe and FeCo were much higher
obvious peroxidase-like activity, owing to the generated
than those of their counterparts, indicating the superior
·OH radicals. On the other hand, no clear catalytic activity
catalytic properties of FeFe and FeCo. A steady-state
could be observed for K4[Fe(CN)6]. Mixing FeIII with
kinetic assay of HRP was also conducted (Fig. S10 in
K4[Fe(CN)6] resulted in the immediate formation of PBNPs.
the ESM). Notably, the Km values of FeFe with both Although the concentration of FeIII ions was reduced
by one half, the formed PBNPs exhibited significantly
Table 1 Comparison of kinetic parameters of as-prepared
higher peroxidase-like activity (Fig. S12(a) in the ESM).
nanomaterials and HRP
In addition, the PBNPs were also found to possess
Km (mM) Vmax (μM·s−1)
Catalysts catalase-like activity, with the catalytic decomposition
TMB H2O2 TMB H2O2
of H2O2 into H2O and O2 [35]. Interestingly, heme was
FeFe 0.359 4.729 1.941 1.737
also responsible for the high activity of native catalase.
CoFe 0.493 4.828 0.120 0.032
On this account, control experiments of catalase mimic
NiFe 0.464 1.041 0.109 0.014
between PBNPs and FeIII ions were conducted. The
CuFe 0.674 2.063 0.149 0.037 decomposition of H2O2 to O2 could be monitored by
FeCo 0.246 3.594 0.474 0.550 following the changes in H2O2 absorbance at 240 nm.
CoCo 0.953 — 0.002 0 The PBNPs showed a markedly high activity (Fig. 5(c)).
NiCo — — 0 0 Gas bubbles could be observed directly after the addition
CuCo 0.202 3.071 0.010 0.006 of PBNPs (Fig. S12(b) in the ESM). The FeNx unit, with
HRP 0.418 3.109 0.360 0.290 intrinsic plane quadrilateral structure, showed catalytic

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6 Nano Res.
Figure 5 (a) Schematic illustration of peroxidase-like and catalase-like activity of PBNPs. (b) Analysis of peroxidase-like activity in
the presence of 2 mM H2O2. (c) Analysis of catalase-like activity in the presence of 20 mM H2O2.
properties similar to those of heme, implying the possible
presence of Fe(IV)=O species in the FeNx unit assembled
by C≡N ligands. However, unlike the Fe-TMAL complexes,
the direct visualization of the Fe(IV)=O intermediate
in PBNPs has many limitations: two different forms of Fe
(e.g., high-spin FeNx and low-spin FeC6) and a low
proportion of active intermediate. Further investigations
of the specific reaction process are needed.
3 Conclusions
In summary, by comparing the catalytic activities of
PBNPs and of a series of PBAs, we demonstrated that
N-coordinated Fe units (FeNx) were responsible for
the peroxidase-like activity of the PBNPs. The intrinsic
heme-like structure endowed the FeNx unit with the
desired enzyme-mimetic activity. In this sense, the PBNPs
represent the first example of a bio-inspired nanozyme.
Furthermore, we discovered that the macrocyclic ligand
assembled by C≡N groups may provide an appropriate
environment to support the Fe(IV)=O species. The
present study is expected to promote the extensive
application of PBNPs in biomimetic reactions.
4 Materials and methods
4.1 Materials
Iron nitrate nonahydrate (Fe(NO3)3·9H2O, 99.99%),
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cobalt nitrate hexahydrate (Co(NO3)2·6H2O, 99.99%),
copper acetate monohydrate (Cu(CH3COO)2·H2O, 99.95%),
nickel acetate tetrahydrate (Ni(CH3COO)2·4H2O, 99.0%),
potassium ferrocyanide trihydrate (K4[Fe(CN)6]·3H2O,
99.5%), potassium ferricyanide (K3[Fe(CN)6], 99.5%),
and citric acid monohydrate (C6H8O7·H2O, 99.5%) were
purchased from Aladdin (Shanghai, China). Potassium
hexacyanocobaltate (K3[Co(CN)6], 98%) was purchased
from J&K Chemical Technology (Beijing, China). TMB
(99%) and HRP (300 U·mg−1) were purchased from
Sigma-Aldrich and Roche, respectively. All chemicals
were used as received, without further purification.
Ultrapure water was used throughout the experiments.
4.2 Characterization
TEM images were obtained with a Hitachi H-8100 EM
microscope operated at 100 kV. SEM images were obtained
with a Hitachi SU-8020 (Japan) field-emission microscope
with an accelerating voltage of 3.0 kV. XRD analysis
was carried out on a D8 Advance (Bruker, Germany)
diffractometer with Cu Kα radiation (λ = 1.5406 Å).
XPS measurements were performed on an ESCALAB-
MKII (VG Co., UK) spectrometer with an Al Kα
excitation source. FT-IR spectra were measured with
a Vertex 70 (Bruker, Germany) spectrometer. UV-vis
absorption measurements were carried out on an
Agilent Cary 60 (Varian) UV-vis-near-infrared (NIR)
spectrometer.
Nano Res.
4.3 Synthesis of PBNPs and PBAs
FeFe was produced according to a previous report. A
0.01 mmol aliquot of K4[Fe(CN)6] and 5 mmol citric
acid were dissolved in 20 mL H2O under stirring at
60 °C (solution A). Another 0.05 mmol aliquot of
Fe(NO3)3·9H2O and 5 mmol citric acid were dissolved
in 20 mL (solution B). Solution B was then added
dropwise into solution A over 10 min and kept under
stirring at 60 °C. The solution turned bright blue
immediately during the mixing process. The reaction
was continued for 5 min and then cooled to room
temperature under stirring for another 30 min. To
obtain FeFe, the mixture was centrifuged at 9,500
rpm for 10 min and then washed three times with
ethanol. Finally, the obtained FeFe product was dried
at 30 °C overnight under vacuum.
CuFe and NiFe were synthesized by a similar procedure,
using Cu(CH3COO)2·H2O or Ni(CH3COO)2·4H2O to
replace Fe(NO3)3·9H2O, and K3[Fe(CN)6] instead of
K4[Fe(CN)6]·3H2O. CoFe was synthesized at room
temperature using Co(NO3)2·6H2O and K3[Fe(CN)6]
as precursors. FeCo, CoCo, NiCo, and CuCo were
synthesized using K3[Co(CN)6] and the corresponding
metal salts. The detailed synthesis conditions are
summarized in Table S1 in the ESM.
4.4 Peroxidase-like activity
In a typical experiment, 950 μL of acetate buffer solution
(0.1 M, pH 4.0), 20 μL of 0.25 mg·mL−1 catalysts, 10 μL of
25 mM TMB, and 20 μL of 100 mM H2O2 were added
sequentially into a 1.5 mL tube. The catalytic oxidation
of TMB was studied by measuring the absorption
changes of the oxidized form of TMB at λmax = 652 nm
( = 39,000 M−1·cm−1). The Michaelis–Menten constant
was calculated using a Lineweaver– Burk plot: 1/ν = Km/Vm
(1/[S] + 1/Km), where ν is the initial velocity, Vm represents
the maximal reaction velocity, and [S] corresponds to
the substrate concentration. All reactions were performed
at room temperature.
Acknowledgements
We are grateful for the support from the National
Natural Science Foundation of China (Nos. 21375123
and 21675151) and the Ministry of Science and Technology
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7
of China (Nos. 2013YQ170585 and 2016YFA0203203).
Electronic Supplementary Material: Supplementary
material (synthesis conditions, additional TEM images,
FTIR spectra, XPS spectra, Lineweaver-Burk plots, and
optical image) is available in the online version of this
article at https://doi.org/10.1007/s12274-018-2079-8.
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