Two New Preyssler-Type Polyoxometalate-Based Coordination Polymers and Their Application in Horseradish Peroxidase Immobilization

Article abstract of DOI:10.1002/chem.201703158

Enzyme immobilization is of increasing importance for biocatalysis, for which good supports are critical. Herein, two new Preyssler-type polyoxometalate (POM)-based coordination polymers, namely, {[Cu(H₂biim)₂][{Cu(H₂biim)

Full text of DOI:10.1002/chem.201703158

A Journal of
Accepted Article
Title: Two new Preyssler-type polyoxometalate-based coordination
polymers and their application in horseradish peroxidase
immobilization
Authors: Jing Du, Mei-Da Cao, Shu-Li Feng, Fang Su, Xiao-Jing Sang,
Lan-Cui Zhang, Wan-Sheng You, Mei Yang, and Zai-Ming
Zhu
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To be cited as: Chem. Eur. J. 10.1002/chem.201703158
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Two new Preyssler-type polyoxometalate-based coordination
polymers and their application in horseradish peroxidase
immobilization
Jing Du, Mei-Da Cao, Shu-Li Feng, Fang Su, Xiao-Jing Sang, Lan-Cui Zhang,* Wan-Sheng You, Mei
Yang* and Zai-Ming Zhu*[a]
Abstract: Enzyme immobilization is of increasing importance for the
biocatalysis, to which the good supports are critical. Here, two new
Preyssler-type polyoxometalate (POM)-based coordination polymers,
catalyzed processes are of great significance for large-scale
industrial applications.[16] However, they are suffering from the
high cost and difficult to recovery and recycle. Generally, these
disadvantages can be overcome by immobilizing enzyme on the
prefabricated supports.[17] Many properties including thermal,
mechanical and chemical stablities and insolubilities of the
employed supports are crucial for a successful immobilization
namely
{[Cu(H2biim)2][(Cu(H2biim)2(μ-
H2O))2Cu(H2biim)(H2O)2]H[((Cu(H2biim)(H2O)2)0.5)2((μ-
C3HN2Cl2)(Cu(H2biim))2)(Z(H2O)P5W30O110)]·xH2O}n (Z = Na, x = 9,
compound 1; Z = Ag, x = 10, compound 2; H2biim = 2,2′-biimidazole)
were designedly synthesized. Compounds 1 and 2 exhibit the same
skeletons which contain multiple Cu(II) complex fragments and penta-
supported {ZP5W30} ({NaP5W30} and {AgP5W30}) clusters. They were
firstly employed for immobilizing horseradish peroxidase (HRP). The
results show that compounds 1 and 2 as good supports for HRP
immobilization exhibit higher enzyme load, lower loading time, and
excellent reusability. The immobilized enzyme (HRP/1 or HRP/2) was
further applied to detect H2O2, and good sensitivity, wide linear range,
low detection limit and fast response were achieved. This work shows
that POM-based hybrid materials are a new kind of promising
supports for enzyme immobilization.
process.
Moreover,
high
loading
capacities,
good
biocompatibilities, low cost and environmentally benign are also
important for a favorable support.[18] POMs are characterized by
highly negative charges, oxygen-rich surfaces and abundant
structures,[19] and more worthy of their unique biological
compatibilities.[20] As a result, POMs may be a new type of support
materials for immobilizing enzymes. Compared with the classical
Keggin- or Dawson-type POMs, Preyssler-type POMs are
promising as candidates owing to the following attributes: firstly,
the Preyssler anions are one of the biggest precursors with high
negative charges, which is of benefit to the electrostatic
interaction between enzymes and POMs; secondly, the Preyssler
anions own abundant exposed surface oxygen atoms (30 oxygen
atoms), which can offer a large number of active sites to link
enzymes through hydrogen bonds; finally, Preyssler-type POM is
very stable both in a wide pH range (1-10) and at higher
temperature (up to 300 °C).[21] However, it is worth noting that the
water solubility of traditional unmodified POMs limited their
applications in enzyme immobilization. So the introduction of
large cations (e.g. organic or metal-organic complex cations) into
the POM skeleton is an efficient method to decreas its solubility.
2,2′-biimidazole (H2biim), as a N-containing bi-heterocyclic ligand,
Introduction
Polyoxometalates (POMs) are a class of nanosized metal-oxygen
clusters, which have been intensively researched in various fields
such as catalysis, optics, energy and materials and so on.[1–7]
Thereinto, in biology POMs are gaining increasing attention
owing to their antiviral, antibacterial and anticancer properties.[8-
10]
POMs are capable of interaction with diverse
biomacromolecules through electrostatic interactions or charge-
charge interactions, hydrogen bonding, covalent bonding and
van-der-Waals interactions.[11-13] For example, Rompel reported
that Na6[TeW6O24]·22H2O can selectively bind to the specific
regions of proteins through covalent bond interactions during
protein crystallization.[14] Wang’s group found that TPPA-PMo12
hybrid material can selectively adsorb and isolate β-lactoglobulin
through electrostatic interactions from milk.[15]
possessing abundant
N atoms and versatile coordination
behaviors, it is a valuable building block for constructing
inorganic-organic hybrid materials with novel structures and
special properties.[22–24] Moreover, the hydrogen bonded 2,2′-
H2biim-TM complexes can be used as a tool of crystal
engineering.[23] Meanwhile, H2biim and its derivatives have good
biological activity, for example, they display antibacterial activity
and imitative behavior as the active center of the zinc hydrolase
enzyme.[25,26] Therefore, combination of Preyssler-type POMs and
H2biim to construct hybrid crystalline materials especially
coordination polymers that possess strong bonding skeletons,
well-defined structures and precise establishment of structure-
property relationships is a potential method to obtain a new kind
of supports for enzyme immobilization.
To the best of our knowledge, POMs are generally dispersed or
supported on other solid supports in POM-based materials, while
POMs as supports for enzyme immobilization have not been
reported. In the present work, the two Preyssler-type POM-based
materials (compounds 1 and 2) were successfully synthesized by
introducing Cu(II) cations and H2biim organic ligand into the
As we know, enzymes are a kind of bioactive proteins with high
efficiency, low pollution and high selectivity, and enzyme-
[a]
J. Du, M. D. Cao, S. L. Feng, F. Su, X. J. Sang, Prof. L. C. Zhang,
Prof. W. S. You, Prof. M. Yang, Prof. Z. M. Zhu
School of Chemistry and Chemical Engineering
Liaoning Normal University
Huanghe Road 850, Dalian 116029, P. R. China
E-mail: zhanglancui@lnnu.edu.cn; yangmeils@163.com;
chemzhu@sina.com.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.2017xxxxx.
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system of Preyssler-type POM {NaP5W30}/{AgP5W30} under
hydrothermal conditions and used as enzyme-immobilizing
supports for the first time. The optimal immboilized conditions of
horseradish peroxidase (HRP) on compound 1 and 2 (i.e. HRP/1
or HRP/2) were researched systematically. The catalytic activity
and recyclability of HRP/1 and HRP/2 were also evaluated.
Results and Discussion
Synthesis. During the past years, our group has successfully
synthesized a series of 3D POM-based inorganic-organic hybrid
materials with TM-H2biim complexes by hydrothermal method.[27–
Scheme 1. Schematic diagram of synthetic route of compound 1/2
30]
These materials revealed salient catalytic properties and
structural stabilities. However, it is still a great challenge to
construct high dimensional coordination polymers by using big
sized POM building blocks. The {ZP5W30} unit with higher
negative charge is very stable in a wide range of pH value, which
is favor of constructing multifunctional stable materials. To
synthesize some new structures to further build up the interaction
with HRP, N-containing heterocyclic ligands such as H2biim and
imidazole were employed, and some copper salts such as
from the resulting solution by slow evaporation of solvents at room
temperature. The green compound was characterized by the
powder X-ray diffraction (PXRD) analysis. It can be seen from the
comparation of the PXRD peaks (Figure S1) between our sample
and the reported result (refer to CCDC 285638[34] ) that the green
sample is simple Cu-coordination complex [Cu(H2biim)Cl2]
(Scheme 2b), and there exits no C–Cl bond, and the C-C bond
between the two imidazole rings is not broken. Hence, it can be
inferred that CuCl2 and {ZP5W30} must promote the breaking of C–
C bond and the C–Cl bond formation, and the C–C bond breakage
may be also caused by the redox-active of the Preyssler-type
POMs.[35, 36] In order to investigate the influence of the center
Na(I)/Ag(I) on the structure of the {ZP5W30}-based product, in the
Cu(NO3)2·3H2O,
CuSO4·5H2O,
Cu(CH3COO)2·H2O,
and
CuCl2·2H2O were selected as TM ion sources. The synthetic route
of compound 1/2 is illustrated in Scheme 1. Initially, when the
mixture of
K12.5Na1.5[NaP5W30O110]·15H2O (abbreviated as
parent-1), Cu(NO3)2·3H2O or CuSO4·5H2O /Cu(CH3COO)2·H2O,
H2biim and H2O was heated at 140 °C for 3 days, no crystalline
materials were discovered (Step Ia). When CuCl2·2H2O was used
in place of the above mentioned Cu(II) compounds under the
same conditions as those of Step Ia, fortunately, compound 1 was
obtained in crystalline form (green strip crystals, Step II). More
interestingly, 4,5-dichloroimidazole (C3HN2Cl2) was unexpectedly
discovered at the same time in the solid structure of compound 1
via X-ray diffraction analysis. Where did the imidazole ring come
from? In order to seek the source of 4,5-dichloroimidazole and
some experiments were designed and carried out as follows: (1)
as shown in Scheme 1, Step Ib, the reaction was performed in
NaCl solution rather than in H2O, which is to provide additional Cl–
ions, and other conditions were the same as described in Step Ia;
(2) on the basis of Step Ib, an appropriate amount of imidazole
was added (Step Ic). Unfortunately, only amorphous precipitates
were obtained in the above experiments.
synthetic
process,
we
used
K14[AgP5W30O110]·18H2O
(abbreviated as parent-2) in place of parent-1, all the above
experiments were repeated under the same conditions. Similarly,
for the starting materials Cu(NO3)2·3H2O, CuSO4·5H2O and
Cu(CH3COO)2·H2O, no crystalline products were obtained, while
for the starting material CuCl2·2H2O, the isostructural compound
2 containing {AgP5W30} unit was easily obtained (Scheme 1, Step
II), which further proves that the presence of CuCl2·2H2O is critical
to formation of compound 1/2.
Taking into account of these summarized experiments, it
seems reasonable to assert that the 4,5-dichloroimidazole group
must derivate from CuCl2·2H2O and H2biim in the present of
Preyssler-POMs (Scheme 2a), while the extraneous Cl– anions
and imidazole molecules may not form 4,5- dichloroimidazole
under our giving conditions. Additionally, In our previously
reported work, many other polyoxometalate-based copper-H2biim
complexes such as Cu-H2biim-{P2Mo5}, Cu-H2biim-{Mo8O26} and
Cu-H2biim-{P2W18} series,[31-33] 4,5-dichloroimidazole was not yet
observed in their synthetic processes. To check if the 4,5-
dichloroimidazole is also formed in the absence of the Preyssler-
POM, some experiments were carried out and repeated under the
similar conditions to those of compound 1. None of other
compounds except green microcrystalline samples was obtained
Scheme 2. Chlorination of H2biim during the synthetic process of compound 1/2
(a); the reaction of CuCl2·2H2O and H2biim in the absence of the Preyssler-
POM (b)
Crystal structures. Single-crystal X-ray diffraction analysis
revealed that compounds 1 and 2 are coordination polymers
composed of Preyssler-type polyoxoanion {ZP5W30} (Z = Na, Ag)
units, Cu(II)-H2biim/H2O complexes and lattice water molecules
(9 and 10 for compounds 1 and 2 respectively) (Figures 1 and S2),
exhibiting similar symmetric structures and the most outstanding
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features: each Preyssler-type polyoxoanion coordinates to five
Cu(II) (four Cu1 and two half-occupancy Cu5) ions forming penta-
supported structure (Figure 1a). Although the Preyssler-type
anion has abundant O atoms to coordinate to metal ions, such a
polyoxoanion with high coordination number was still hard to
obtain, and multi-supported polyoxoanion could play an important
role in the stability of the solid structures. In general, the centers
of most of Preyssler-type polyoxoanions contain two sites, in
compound 1 or 2, the two Na+ or Ag+ sites have the same
occupancy of 50% (see Figure S2a-b). The introduction of Ag(I)
and Cu(II) ions into compound 2 represents two kinds of TM ions
modified Preyssler-type POM-based inorganic-organic hybrid
material. As shown in Figure 1b–e, the five crystallographic-
separate Cu(II) centers with multiple coordination modes formed
mono-, bi- and tri-nuclear Cu(II) complex fragments in the two
compounds. In addition, all H2biim molecules act as bidentate
ligands coordinating with Cu(II) ions. As shown in Figure 1e, the
two Cu1 atoms are bridged by 4,5-dichloroimidazole, forming a
binuclear complex. Cu1 is penta-coordinated by two N atoms from
one H2biim molecule, one N atom from a 4,5-dichloroimidazole
fragment and two O atoms from two {ZP5W30} units, forming a
square-pyramidal CuN2O3 configuration. The bond lengths of
Cu1–N and Cu1–O are in the ranges of 1.938(18)–1.989(17) Å
and 2.033(14)–2.337(16) Å for compound 1, and 1.947(17)–
2.003(16) Å and 2.020(12)–2.342(14) Å for compound 2 (see
Tables S1 and S2). As seen from Figure 1b, two Cu2 centers are
bridged by one Cu3 center via two μ-H2O (O7W) ligands, forming
an isolated tri-nuclear complex fragment. Each Cu2 atom adopts
a square-pyramidal CuN4O (four N atoms of two H2biim molecules,
one μ-H2O ligand) coordination mode, and Cu3 atom adopts an
octahedral CuN2O4 (two N atoms from one H2biim molecule, two
μ- and two terminal H2O ligands) coordination mode. The bond
lengths of Cu3–N and Cu3–O are in the normal range except the
two longer bonds of Cu3–O7W (about 2.7 Å).[37] Cu4 is the center
of a quadrilateral mono-nuclear complex fragment isolated
around the {ZP5W30} cluster, and it coordinates with four N atoms
from two H2biim molecules with Cu4–N distances of 2.02(3)–
2.10(3) Å for compound 1 and 1.99(3)–2.07(2) Å for compound 2
(Figure 1c, Tables S1 and S2). Each Cu5 atom bonds to one
H2biim ligand, one terminal O atom and two aqua ligands forming
a square-pyramidal mono-nuclear Cu(II) complex subunit (Figure
1d). The Cu5–N and Cu5–O bond lengths are 2.000(10)–
2.344(10) Å and 1.949(10)–2.22(5) Å for compound 1, and
1.95(4)–2.155(10) Å and 1.993(10)–2.30(4) Å for compound 2.
The W−O distances are in the normal ranges of 1.683(16)–
2.287(14) and 1.693(13)–2.292(13) Å for compounds 1 and 2,
respectively (Tables S1 and S2). The bond valence sum (BVS)
calculations show that the oxidation states for all W, P, Ag and Cu
atoms are +6, +5, +1 and +2, respectively.[38]
Figure 1. The hexa-supported polyoxoanion structure in compound 1/2 (a); the
Cu complex units of compound 1/2 (b–e); the 1D chain viewed along c axis in
1/2 (f). All crystal water molecules and H atoms are omitted for clarity
S6, Tables S3 and S4) and the electrostatic attractions among
complex cations and polyoxometalate anions. It is obviously that
the 4,5-dichloroimidazole fragment plays a vital role in the crystal
packing. Simultaneously, the strong hydrogen bonds are very
beneficial to the stability of the structure and the interaction with
enzyme.
Characterization of compounds 1 and 2. The IR spectra of
compounds 1 and 2 (presented in Figure S7) all show the
characteristic vibrations of the {ZP5W30} polyoxoanion compared
with those of parent-1 and parent-2 (Figure S8): the P–O, W–
O
terminal and W–Obridging vibrations are at 1168–1087 cm–1, 925 and
40]
921cm–1, and 786 and 798 cm–1, respectively.[39,
The wide
frequency range of 3464–2920 cm–1 is associated to ν(O–H) of
water molecules, ν(C–H) and ν(N–H) of H2biim molecules. The
weak peaks in the region of 1631–1411 cm–1 can be assigned to
the imidazole ring stretching.[27,
To verify the purity of the
28]
products, the PXRD patterns of compounds 1 and 2 were
recorded and presented in Figure S9, the simulated patterns of
compounds 1 and 2 were calculated using CIF file by Mercury
software (Copyright Cambridge Crystallographic Data Centre). As
seen from Figure S9a–b, the characteristic diffraction peaks of
compounds 1 and 2 are all at 5.21 (100), 5.59 (002), 5.87(110),
6.50 (111), 9.98 (023), 10.24 (113), 11.15 (132), 15.92 (310),
16.16 (311), 22.45 (082), 24.51 (157), 25.68 (192), 25.87 (425)
and 27.62° (048), which indicated that compounds 1 and 2 are
isostructural. Furthermore, the experimental data match well with
the simulated data at the positions of main diffraction peaks,
indicating the crystalline products are in the pure phase.[41] The
electrochemical property of compound 1/2 by using compound
1/2 bulk-modified carbon paste electrode (i.e. 1/2-CPE) was
carried out. As shown in Figure S10, 1/2-CPE and parent-1 show
similar redox peaks, indicating that the {ZP5W30} units were
successfully introduced into the compounds 1 and 2. The
photoluminescence properties of parent-1, H2biim, compounds 1
and 2 in the solid state at room temperature were shown in Figure
S11. Parent-1, compounds 1 and 2 all display luminescence with
three emission peaks at 433, 450 and 470 nm (λex= 280 nm),
More interestingly, as shown in Figures 1f and S3, the adjacent
Preyssler-type
anions
are
bridged
by
[(μ–
C3HN2Cl2)(Cu(H2biim))2]3+ complex cations into infinite 1D zigzag
chains, and further packed into a 3D supramolecular structure
(see Figure S4) along with the Cu2, Cu3 and Cu4–H2biim
complex fragments through extensive hydrogen bonding
interactions (N—H···O/OW 1.90–2.66 Å and 1.82–2.67 Å for
compound 1 and compound 2, respectively, see Figures S5 and
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which should be assigned to LMCT (O→W).[42] The emission peak
at 345 nm of H2biim should be assigned to π*→π transition. The
emission peaks at 400 and 415 nm of compounds 1 and 2 may
be caused by the coordination of Cu2+ and H2biim leading to
LMCT (N→Cu).[43] This indicates that {ZP5W30} unit and Cu-
H2biim complex were successfully introduced into the two
compounds.
Meanwhile, it can be seen from Figure 2b that the TOF of
HRP/1 gradually increases with the pH increasing from 3.5 to 4.5,
while decreases from pH 4.5 to 8.5. Comprehensive
consideration of the enzyme loading and the immobilized enzyme
activity, the optimal pH of enzyme immobilization is 4.5.
Furthermore, the effects of the optimum immobilized time and
enzyme concentration in the medium on the enzyme loading were
also studied. As shown in Figure S18, the enzyme loading
increases with the immobilized time and HRP concentration
increasing, and the loading capacity of the enzyme on compound
1 can reach the maximum at 120 min and 1.6 mg mL–1 of enzyme
concentration. Summing up the above-mentioned results, the
optimum conditions for enzyme immobilization were as follow: the
HRP concentration was 1.6 mg mL–1 in a phosphate buffer
solution (PBS, 0.1 mol L–1) with pH 4.5, and the immobilized time
was 120 min. The enzyme loading was calculated as follows:
To further confirm the crystal structures and thermal stabilities,
the thermogravimetric (TG) curves of compounds 1 and 2 were
also researched. As shown in Figure S12, there are two-step
weight losses in the temperature range from 35 to 1000 °C. The
first weight loss at 35–200 °C is 3.01 % (calcd. 2.98 %) for
compound 1 and 2.89 % (calcd. 3.14 %) for compound 2, which
can be ascribed to the loss of all water molecules. The second
weight losses from 200 to 1000 °C are 16.88 % (calcd. 18.96 %)
and 17.44 % (calcd. 18.80 %) for compounds 1 and 2,
respectively, which are attributed to the losses of all organic
composites and partial sublimable phosphorus oxide species.[44]
The above results are also verified by the IR spectra of
compounds 1 and 2 suffering from heating at 200, 450 and 800 °C
in air (Figure S13). Indeed, the characteristic absorption bands of
H2biim disappeared after 450 °C, and the characteristic νas(P–O)
of {ZP5W30} polyanion at 1168–1087 cm–1 are weaken after
heating at 800 °C. This indicates that compounds 1 and 2 were
stable up to 200 °C.
Enzyme immobilization and enzymatic activity. Firstly, as the
support for enzyme immobilization, the stability of the support is
more important in higher pH solutions. As seen from Figure S14,
compound 1 is sable at pH 7–9. In the range of pH 3.5–7.5, the
effect of pH on the enzyme loading was evaluated (Figure 2a). it
is found that the enzyme loading gradually decrease with the pH
increasing which may due to the existence of electrostatic
interaction between compound 1/2 and HRP. in order to confirm
the electrostatic interaction, the surface charges of the sole HRP,
pure compound 1 and compound 1 loaded with different
concentrations of HRP were analyzed by the Zeta potential
(Figures S15 and S16).[45] As seen from Figure S15, with the pH
increasing, the positive potential of the free HRP decreases, while
the negative potential of compound 1 almost unchanged in pH
range from 3.5 to 8.5, so it can be deduced that the electrostatic
interaction between HRP and compound 1 is the main binding
force, and the decrease of enzyme loading is due to the
decreased positive charge of HRP in the pH from 3.5 to 8.5. As
seen from Figure S16, the Zeta potentials are gradually increasing
with the enzyme loading increased, due to the negative charge of
the POM-based compounds was neutralized by the positive
charge of the HRP molecules, this also further illustrates the
interaction between HRP and POM-based compound was
electrostatic interaction.[45] Meanwhile, several works suggest the
frequent interactions between POMs and biomacromolecules are
electrostatic interactions/charge-charge interactions, hydrogen
bonds, covalent bonds and van-der-Waals interactions.[12, 46–48]
From the point of the literatures, the above experimental results
and the crystal structures of compounds 1 and 2, we speculate
that the interactions between compound 1 or 2 and protein side
chains in HRP may have electrostatic interaction, hydrogen bond
and van-der-Waals interaction (see Figure S17).
Enzyme loading (mg g–1) = (c0– c) V/ Ws
(1)
Where c0 and c are the initial and final HRP concentrations in
the immobilization medium solution (mg mL–1), respectively, they
can be gained from the standard curve of HRP. V is the volume
of the enzyme solution (mL), and Ws represents the amount of the
support (compound 1/2, g).[49]
According to equation (1) and Figure S19, under the optimum
conditions, the maximum of enzyme loadings are reached to
158.7 and 157.5 mg g–1 for compounds 1 and 2, respectively (see
Figure S20). Compared with the reported materials for HRP
immobilization, such as periodic mesoporous organosilica (126
mg g–1), nanoporous copper (8.8 mg g–1), modified chitosan
(82.66 mg g–1) and graphene oxide (0.7 mg g–1),[50–53] compounds
1 and 2 harvested higher loading capacity of HRP at shorter
immobilizing time in this paper. The detail immobilizing conditions
of theses supports are listed in table S6.
Figure 2. The enzyme loading (a) and the immobilized enzyme activity (b) for
compound 1 at different pH values (HRP solution: 1.6 mg mL–1, compound 1:5
mg, the immobilized time: 120 min, in PBS, the line is drawn for guiding the eye
only)
To further confirm the absorption of HRP on compound 1/2,
HRP was labeled with Rhodamine B isothiocyanate (RhBTC), and
subjected to the same immobilization procedure under the
optimum condition, then the RhBTC-labeled HRP/1 or HRP/2 was
test by Confocal Laser Scanning Microscopy (CLSM) images.[54]
From the results of CLSM as shown in Figure 3a–b, it can be
observed that RhBTC-HRP (red) actually presented in compound
1 or 2. In addition, the TGA of HRP/1 (2.94 mg HRP/1, HRP
loading: 158.7 mg g–1) or HRP/2 (2.18 mg HRP/2, HRP loading:
157.5 mg g–1) in air also confirmed the presence of HRP in the
composite (Figures 3c–d, S21). As seen from Figures 3c–d and
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S21, the first decomposition stage of compound 1 or compound 2
and HRP/1 or HRP/2 exhibited a gradual weight-loss of 3% up to
ca. 200 °C, which can be ascribed to the loss of all water
molecules. The second decomposition stage of the HRP/1 or
HRP/2 started from 200 and finished about 600 °C, the pure
compound 1 or compound 2 had much less weight loss during this
temperature range. About extra 12.95 % and 12.45% of weight
losses occurred during the second stage for HRP/1 and HRP/2,
respectively, which can be attributed to the decomposition of HRP
molecules.[54] The HRP loadings of HRP/1 and HRP/2 were 13.70
and 13.61 wt% calculated from equation (1), respectively, which
agreed well with the weight loss in the TGA curves. The results of
the CLSM image and TGA showed that HRP is immobilized on
compound 1 or compound 2.
for 15 min, it is found that the activities of them were no significant
decline before 40°C. When the temperature reached 50 °C, their
activities all began to decline significantly. The results suggest the
maximum tolerated temperature of HRP/1 or HRP/2 was 40 °C.
The operative stabilities of the free HRP, HRP/1 and HRP/2 were
als evaluated at 40 °C. Figure S24b shows the activities of free
HRP and HRP/1 or HRP/2 in 100 min at 40 °C, which indicated
that although the activity of HRP/1 or HRP/2 decreased slightly
compared with free HRP, most of their activities remained. Figure
S25 illustrated that the activities of both HRP/1 or HRP/2 and the
free HRP were gradually decreased after incubating for 10 days,
and this indicates that the decrease of enzyme activity is not the
influence of the POM-based material. Meanwhile, by referring to
several earlier literatures that the covalent immobilization of
enzymes on supports resulted in the stabilization of enzymes
against various forms of denaturation,[58] the thermal and storage
stabilities of HRP/1 or HRP/2 against various forms of
denaturation were no obviously improved, which could be due to
the interaction existed in the immobilized HRP was mainly
electrostatic interaction rather than covalent binding
In addition, the most outstanding advantage of the enzyme
immobilization on POM supports is that the immobilized enzyme
can be easily separated from the reaction system and used
repeatedly. As shown in Figure 4, after recycling for 12 times, the
activity of the HRP/1 or HRP/2 decreased, but 50% of initial
activity was retained after recycling for 10 times. The possible
reasons caused the decreased activity might be due to some
leaching during the repeated washing and a long time contact with
high concentrations of substrate.[51] Compared with the
immobilized HRP reported in the literatures, the reusability of
HRP/1 or HRP/2 was better than those of the immobilized
enzymes through pore adsorption and surface adsorption,[50,51]
and worse than those of the immobilized HRP binded covalently,
which due to the electrostatic interaction between the POMs and
HRP.
Figure 3. CLSM images of RhBTC-labeled HRP/1 (a) and HRP/2 (b) (red),
excitation at 543 nm. The TGAs of compound 1 (c) and compound 2 (d) before
and after enzyme immobilization
To investigate the structural stability of the title crystalline
materials as enzyme immobilization supports, the PXRD patterns
and IR spectra of compounds 1 and 2 before and after enzyme
immobilization were tested and shown in Figures S22 and S23.
As seen from Figure S22 the main diffraction peak positions are
consistent with the simulated values of compounds 1 and 2,
respectively, which indicated the structure of compound 1 or 2
was preserved. On the other hand, it also reflected that HRP was
only adsorbed on the surface of compound 1 or 2, did not enter
into the crystal lattices of the supports. As shown in Figure S23,
the IR characteristic peaks of the powder samples after
immobilization are consistent with those of crystalline compounds
1 and 2 before immobilization. These results also prove that the
structures of compounds 1 and 2 are stable and efficient support
materials for enzyme immobilization.
The thermal stabilities of the free HRP and HRP/1 or HRP/2
were studied between 20 and 60 °C, and their operative and
storage stabilities were researched by incubating them for 0-100
min at 40 °C and 1-10 days at room temperature (ca. 25°C),
respectively. The activities of these materials under different
conditions were determined by Worthington method at pH 4.5. [55–
Figure 4. The activity of HRP/1 or HRP/2 after recycling for 12 times
A colorimetric system involving that HRP catalyzed the co-
oxidation of phenol and 4-aminoantipyrine (4-AAP) was
developed for the determination of H2O2 concentration.[59] As
57]
As shown in Figure S24a, when the free HRP and HRP/1 or
HRP/2 were subjected to different temperatures from 20 to 60 °C
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shown in Figure 5, for HRP/1 (HRP loading: 158.7 mg g–1) or
HRP/2 (HRP loading: 157.5 mg g–1), the concentration of H2O2 as
low as 1.61 × 10–6 and 1.49 × 10–6 mol L–1 can be detected with
the linear ranges of 1.61 × 10–6 – 3.0 × 10–4 and 1.49 × 10–6 – 3.0
× 10–4 mol L–1, respectively (see the insets in Figure 5a-b). These
results also confirmed that the activity of HRP is still remained
after immobilized on compound 1/2, and they are a kind of
potential materials for H2O2 detection.
grade and used without further purification, double distilled water was used
in the enzyme related experiments.
Instrumentations.
Infrared (IR) spectra were obtained on a Bruker AXS TENSOR-27 FT-IR
spectrometer with KBr pellets in the range of 4000–400 cm–1. C, H and N
elemental analyses were performed by a VarioElcube elemental analyser;
P, Cl, W, Cu, Na, Ag were acquired using a Prodigy XP emission
spectrometer. TG analyses were performed on a Pyris Diamond TG-DTA
thermal analyser at a heating rate of 10 °C min–1 from 35 to 1000 °C in air
atmosphere. PXRD data were collected on a Bruker AXS D8 Advance
diffractometer using Cu Kα radiation (λ = 1.5418 Å) in the 2θ range of 5–
60° with a step size of 0.02°. Single crystal X-ray diffraction data was
collected on a Bruker Smart APEX II X-diffractometer equipped with
graphite monochromated Mo Kα radiation (λ
photoluminescence property was determined on
=
0.71073 Å). The
Hitachi F-7000
a
fluorescence spectrophotometer in the solid state at room temperature.
The liquid UV-visible spectra (200–800 nm) were recorded on a Perkin-
Elmer Lambda 35 UV-Visible spectrophotometer. Nuclear magnetic
resonance (NMR) spectra were recorded at room temperature on a Bruker
Avance 500 MHz NMR spectrometer. An inner tube containing D2O was
used as an instrumental lock. Zeta potential was measured at room
Figure 5 The linear calibration plot for H2O2 detection using HRP/1 (a, HRP
loading: 158.7 mg g–1) or HRP/2 (b, HRP loading: 157.5 mg g–1). ∆A = A (the
immobilized HRP, 525 nm) – A (blank, 525 nm). The reaction time is 5 min.
temperature on
potentiometer. All electrochemical measurements were carried out on a
CHI 604B electrochemical workstation at room temperature.
a Malvern Zetasizer Nano ZS90 nano particle
Conclusions
A
conventional three electrode system was used. The working electrode was
1/2-CPE, prepared by hand-mixing of graphite powder with paraffin oil at
a ratio of 3:50 in an agate mortar, Ag/AgCl was used as a reference
electrode, and a platinum wire was used as a counter electrode. CLSM
images were taken on a Zeiss LSM 710 confocal microscope, the detection
wavelength was 543 nm for RhBTC.
In summary, two new Preyssler-type POM-based crystalline
materials have been successfully synthesized through
hydrothermal method. Compounds 1 and
2 revealed the
interesting 3D crystal structures and high thermal stabilities. Most
importantly, the POM-based crystalline materials were firstly used
as supports for the immobilization of HRP, which resulted in a high
immobilized loading of 158.7 and 157.5 mg g–1 for HRP/1 or
HRP/2. The activity of HRP/1 or HRP/2 was remained after
recycling for 12 times. In addition, HRP/1 or HRP/2 was utilized to
detect trace H2O2 with a linear response range of 1.61 × 10–6– 3.0
× 10–4 and 1.49 × 10–6 – 3.0 × 10–4 mol L–1 with a limit of detection
of 1.61 × 10–6 and 1.49 × 10–6 mol L–1, respectively. Therefore, the
Preyssler-type POM-based crystalline materials are potential
candidates for enzyme immobilization supports. The present work
has broadened our research field of POM-based inorganic-
organic hybrid materials. Our further work is already in progress
to synthesize other type POM-based enzyme immobilization
materials.
Preparation of POM-based crystalline materials.
Parent-2. In the synthetic experiment, parent-1 (1.000 g, 0.121 mmol) was
dissolved in 15 mL of double distilled water, and AgNO3 (0.040 g, 0.240
mmol) was added to this solution under stirring. The resulting mixture was
transferred into a Teflon-lined autoclave (30 mL) and kept at 160 °C for 3
days, the transparent liquid was obtained after slow cooling to room
temperature. Then, 1.000 g of solid KNO3 was added into the above mixed
liquid, the resulting precipitate was collected and dissolved in hot water (10
mL), the colorless block crystals of parent-2 (0.75 g) formed via gradually
cooling, and were filtered and dried in air. Elemental analysis calcd (%) for
K14H36O128AgP5W30 (parent-2): H, 0.43; K, 6.51; Ag, 1.28; P, 1.84; W,
65.58. Found (%): H, 0.42; K, 6.47; Ag, 1.22; P, 1.84; W, 65.83.FT IR
(Figure S8): 3460 (s), 2931 (w), 1620 (s), 1168 (m), 1.087 (w), 933 (m),
767 (s) cm–1 31P NMR (Figure S26): δ=–10.29 ppm.
.
Compound 1. A mixture of parent-1 (0.200 g, 0.024 mmol), CuCl2·2H2O
(0.087 g, 0.510 mmol), H2biim (0.028 g, 0.130 mmol) and distilled water
(10 mL) was stirred for 1.0 h. The resulting suspension was sealed in a
Teflon-lined autoclave (30 mL) and kept at 140 °C for 3 days. After slow
cooling to room temperature, green strip crystals were obtained and
washed with distilled water and dried at room temperature to give a yield
of 52% based on W. Elemental analysis calcd (%) for
C63H94Cl2Cu7N42NaO126P5W30 (compound 1): C, 7.83; H, 0.98; N, 6.09; Cl,
0.73; P, 1.60; Na, 0.24; Cu, 4.60; W, 57.07. Found (%): C, 7.63; H, 1.12;
N, 6.23; Cl, 0.85; P, 1.52; Na, 0.31; Cu, 4.33; W, 57.25. FT IR: 3542 (m),
3271 (w), 3136 (w), 2920 (w), 1627 (m), 1519 (w), 1415 (w), 1168 (m),
Experimental Section
Chemicals and enzymes.
HRP (isoenzyme C, product P105528, lot K1506050, E. C. 1.11.1.7, 300
U mg–1, RZ> 3.0, Mw ≈ 40 kDa), 4-AAP and RhBTC were purchased from
Aladdin bio-chem technology Co. Ltd, China. PBS (0.1 mol L–1) with
different pH values (3.5–8.5) were prepared using Na2HPO4, NaH2PO4 or
H3PO4. The RhBTC-labeled HRP was prepared as previously described
and free RhBTC was removed via dialysis against double distilled water.[54]
Dialysis bag (MWCO: 3500) was purchased from OAMAY corporation.
Parent-1 was synthesized and characterized by IR spectroscopy
according to the reported method.[39] All other reagents were analytical
1091 (w), 925 (m), 786 (s) cm–1
.
Compound 2. The synthetic method of compound 1 was applied except
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10.1002/chem.201703158
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replacing parent-1 with parent-2 under the same conditions, and green
strip crystals can also be obtained. Yield: 47 % based on W. Elemental
analysis calcd (%) for C63H96AgCl2Cu7N42O127P5W30 (compound 2): C,
7.75; H, 0.99; N, 6.02; Cl, 0.73; P, 1.59; Cu, 4.55; Ag, 1.10; W, 56.47.
Found (%): C, 7.64; H, 1.21; N, 5.95; Cl, 0.72; P, 1.74; Cu, 4.63; Ag, 1.27;
W, 56.56. FT IR: 3464 (m), 3271 (w), 3151 (w), 2920 (w), 1631 (m), 1535
In addition, the effect of H2O2 concentration on the above model reaction
catalyzed by immobilized enzyme were discussed in detail according to
the reference’s method.[59] The evaluation experiments are as follows: 0.5
mg of HRP/1 (enzyme loading: 158.7 mg g–1) or HRP/2 (enzyme loading:
157.5 mg g–1) was added into 1000 μL of PBS (0.1 mol L–1, pH4.5)
containing H2O2 with different concentration (0.05–0.6 mmol L–1), 4 mmol
L–1 4-AAP and 1 mmol L–1 phenol. The result solution was incubated for 5
min and centrifuged for separating at room temperature. Afterward, the
absorbance of 525 nm was acquired under UV–Vis spectra. Pure
compound 1 or 2 without any HRP was used according to the methods
mentioned above to carry out control measurements to obtain a blank
value.
(w), 1411 (w), 1168 (m), 1087 (w), 921 (m), 798 (s) cm–1
.
Structure determination.
The structures were solved by direct methods and refined by the full-matrix
least-squares fitting on F2 using SHELXTL-97 package.[60,61] Cell
parameters were obtained by the global refinement of the positions of all
collected reflections. All the non-hydrogen atoms were refined
anisotropically. H atoms on C or N atoms were added in calculated
positions. Crystal data and structure refinement parameters of compounds
1 and 2 are listed in Table S5. No protonated O atom was noted in the
polyanion by bond valence sum (BVS) calculations.[37] All H atoms on
water molecules were directly included in the final molecular formula.
Selected bond lengths and angles are listed in Tables S1 and S2.
Hydrogen bonds are listed in Tables S3 and S4. The CCDC reference
numbers are 1429454 and 1429455.
Acknowledgements
This work was financially supported by the National College
Students Innovation and Entrepreneurship Training Program (no.
201510165039), the Provincial Undergraduate Innovation and
Entrepreneurship Training Program of Liaoning (no.
201610165000022), the National Natural Science Foundation of
China (nos. 21671091, 21503103 and 21573099), and Youth
scientific research project of Liaoning Normal University (nos.
LS2015L008 and LS2014L018).
Immobilization of HRP. Compounds 1 and 2 were used as supports for
HRP immobilization. In order to obtain the optimal enzyme immobilization
conditions, the influences of pH, enzyme concentration and immobilization
time on enzyme loading were investigated. With reference to the reported
method, the enzyme was immobilized by adsorption method.[62] The
immobilization of HRP was carried out as follow: 5.0 mg of compound 1/2
powder (the average particle size was ca. 800 nm after grinding, Figure
S27) was added to 500 μL PBS (0.1 mol L–1, pH from 3.5 to 8.5) solution
containing a certain concentration of HRP (0.1 to 2.7 mg mL–1), and the
mixture was gently stirred for a certain time (5 to 120 min) at room
temperature. Then the resulting suspension was centrifuged, and UV-vis
absorption spectrum of the supernatant was recorded, meanwhile, the
precipitate was collected and rinsed for three times with PBS to remove
non-specifically adsorbed enzymes, and thus HRP/1 or HRP/2 was
obtained.
Keywords: polyoxometalates•coordination polymer•crystal
growth• enzyme immobilization• horseradish peroxidase
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Entry for the Table of Contents (Please choose one layout)
Layout 2:
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Jing Du, Mei-Da Cao,Shu-Li Feng, Fang
Su, Xiao-Jing Sang, Lan-Cui Zhang,*
Wan-Sheng You, Mei Yang* and Zai-
Ming Zhu*
Page No.1 – Page No.9
Two new Preyssler-type
Two new Preyssler–type polyoxometalate–based coordination polymers were
synthesized and firstly applied to immobilize horseradish peroxidase (HRP), and
exhibited higher enzyme load, lower loading time, excellent reusability.
polyoxometalate-based coordination
polymers and their application in
horseradish peroxidase
immobilization
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