Cascade-Amplified Time-Resolved Fluorescent Assay Driven by an Enzyme-Integrated Catalytic Compartment as an Artificial Multi-Enzyme Complex

Article abstract of DOI:10.1002/chem.201902222

We here report a simple and efficient strategy of fabricating artificial multi-enzyme complex (MEC) based on the integration of natural enzyme with catalytic compartment. As a proof of concept, this strategy was demonstrated by selecting cholesterol oxidase (ChOx) and Ce^III-based nanoscale coordination polymer (Ce-NCP) with peroxidase-like activity as the models, which forms ChOx@Ce-NCP. Benefitting from the confinement and sheltering effects of Ce-NCP, superior cascade activity and stability in harsh environments were achieved in ChOx@Ce-NCP. Meanwhile, the distinct advantage of ChOx@Ce-NCP has also been highlighted by its negligible substrate inhibition effect and adjustable mass ratio of building blocks. Upon the doping of Tb^III in ChOx@Ce-NCP, a luminescent artificial MEC (ChOx@Ce-NCP:Tb) was further fabricated to drive a cascade amplified time-resolved fluorescent assay within a confined space, showing high sensitivity and specificity toward cholesterol.

Full text of DOI:10.1002/chem.201902222

A Journal of
Accepted Article
Title: Cascade amplified time-resolved fluorescent assay driven by
enzyme-integrated catalytic compartment as an artificial multi-
enzyme complex
Authors: Jie Gao, Caihong Wang, Jinhong Wang, and Hongliang Tan
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Eur. J. 10.1002/chem.201902222
Link to VoR: http://dx.doi.org/10.1002/chem.201902222
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COMMUNICATION
Cascade amplified time-resolved fluorescent assay driven by
enzyme-integrated catalytic compartment as an artificial multi-
enzyme complex
Jie Gao#, Caihong Wang#, Jinhong Wang, and Hongliang Tan* [a]
Abstract: We here report a simple and efficient strategy of fabricating
artificial multi-enzyme complex (MEC) based on the integration of
natural enzyme with catalytic compartment. As a proof of concept, this
strategy was demonstrated by selecting cholesterol oxidase (ChOx)
and Ce(III)-based nanoscale coordination polymer (Ce-NCP) with
peroxidase-like activity as the models, which forms ChOx@Ce-NCP.
Benefiting from the confinement and sheltering effects of Ce-NCP,
superior cascade activity and stability in harsh environments were
achieved in ChOx@Ce-NCP. Meanwhile, the distinct advantage of
ChOx@Ce-NCP has also been highlighted by its negligible substrate
inhibition effect and adjustable mass ratio of building blocks. Upon the
doping of Tb(III) in ChOx@Ce-NCP, a luminescent artificial MEC
(ChOx@Ce-NCP:Tb) was further fabricated to drive a cascade
amplified time-resolved fluorescent assay within a confined space,
showing high sensitivity and specificity toward cholesterol.
catalytic features [7]. Particularly, tremendous interests have
recently been aroused in the fabrication of hybrid catalyst by
coupling nanozyme with natural enzyme [8]. This is because such
hybrid catalyst endows nanozyme with the ability to specially
recognize substrates while eliminating the substrate inhibition
effect of natural enzyme. Within this point, the hybrid catalyst
might be an ideal artificial MEC for driving cascade reactions.
However, the cascade reactions catalyzed by such hybrid catalyst
were mostly performed in a bulk solution but not a confined space,
and thereby only a low overturn was obtained due to the diffusion
losses of intermediates during transportation. Additionally, the
fabrications of the hybrid catalysts are often complicated and low
efficiency as the requirement of covalent binding between natural
enzyme and nanozyme [9]. A solution to the issues may be offered
by co-encapsulating natural enzyme and nanozyme into one
compartment. However, the mass ratios of these two catalysts are
technically difficult to be precisely controlled as natural MEC in
the process of encapsulation.
Herein, we present a new strategy to fabricate artificial MEC by
integrating natural enzyme with catalytic compartment. Different
from conventional compartments that used only for enzyme
encapsulation, the catalytic compartment plays two roles of host
and catalyst, which act synergistically to drive cascade reactions.
As a host, the compartment offers a confined space to allow
effective transportation of intermediates with minimized diffusion
losses while protecting loaded enzymes against harsh
environments. When it performed as a catalyst, the compartment
involves in the cascade reaction and eliminates the substrate
inhibition effect to ensure product generation with high efficiency.
In addition, the artificial MEC is essentially a hybrid catalyst of
natural enzyme and nanozyme and its formation only involves in
In biological system, multi-enzyme complex (MEC) is a highly
ordered assembly of multiple enzymes and involves in a major
class of chemical transformation for mediating signal
transductions and metabolic pathways [1]. Unlike free-floating
enzymes, MEC enables intermediates to be effectively
transported between the active sites on enzyme subunits without
leaving the MEC, resulting in a high local concentration of
intermediates to promote overall catalytic efficiencies toward
desired pathways [2]. Inspired by nature, significant efforts have
been devoted to building artificial MEC [3]. The most common
approach is to confine multiple natural enzymes together within a
nanoscaled compartment [4]. Although encourage results have
been achieved by such approach, the involvement of multiple
natural enzymes also brings it several drawbacks. For example,
to tolerate the various properties of different enzymes, an
appropriate compartment has to be designed and prepared very
carefully [5]. This makes the fabrication process of artificial MEC
complicated and laborious. Moreover, as a trade-off of high
loading efficiency, the overall turnover of artificial MEC is often
limited by the substrate inhibition effect that caused by excess
intermediates in the confined space. A typical example of such
effect is the irreversible inactivation of horseradish peroxidase
(HRP) at high local concentration of H2O2, which is often found in
the encapsulation of single enzyme, and thereby
a high
encapsulation efficiency and adjustable mass ratio of the cascade
catalysts are expected as natural MEC.
To demonstrate this strategy, nanoscale coordination polymer
(NCP) may be an ideal candidate of catalytic compartments due
to its well demonstrated advantages in hosting and mimicking
natural enzymes as compared to conventional hosts and
nanozymes [10]. Nevertheless, to our knowledge, the role of NCP
has been only limited to either a stabilizing host scaffold or an
enzyme mimic till now, whereas the investigations on combining
these two merits into one composite for driving cascade reaction
remain unexplored.
the artificial MEC coupled glucose oxidase with HRP [6]
.
As a fascinating artificial enzyme, nanozyme has been
extensively investigated in a variety of fields due to its unique
As a proof of the concept, Ce(III)-based NCP (Ce-NCP) with
peroxidase-like activity was selected as a model to integrate
cholesterol oxidase (ChOx) for fabricating the artificial MEC,
denonted as ChOx@Ce-NCP. Adenosine triphosphate (ATP) was
used as a bridge linker to synthesize the Ce-NCP because of its
good water solubility and biocompatibility. Figure S1 shows that
the Ce-NCP is spheroid with diameter ranging from 10 to 30 nm
and its formation is originated from the chemical coordination of
Ce3+ with the phosphate and nucleobase moieties of ATP [11]. The
[a]
Jie Gao, Caihong Wang, Jinhong Wang, and Hongliang Tan
Key Laboratory of Chemical Biology of Jiangxi Province
College of Chemistry and Chemical Engineering
Jiangxi Normal University
Nanchang, 330022, China
E-mail: hltan@jxnu.edu.cn
[#] Jie Gao and Caihong Wang contributed equally to this work.
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201902222
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COMMUNICATION
powder XRD pattern reveals that the Ce-NCP is structurally
amorphous (Fig. S2a). From the N2 isotherm in Fig. S2b, we can
see that the Ce-NCP has a BET surface area of 486.37 m2 g-1 and
an average pore size of 2.16 nm, indicating its porosity nature.
The peroxidase-like activity of Ce-NCP was demonstrated by
typical TMB colorimetric assay (Fig. S3) and its highest activity
was found at pH 6.0 (Fig. S4). Based on the fluorescent assay of
terephthalic acid and electron spin resonance spectrum (Fig. S5),
the catalytic mechanism of the Ce-NCP can be ascribed to its
ChOx@Ce-NCP. To confirm this speculation, the absorption
spectra of TMB solutions with Chol were measured at different
conditions. As depicted in Fig. 2a, only the addition of ChOx@Ce-
NCP can cause a typical oxTMB absorption spectrum, while
almost no oxTMB absorbance was recorded in the presence of
individual ChOx or Ce-NCP. This indicates that TMB oxidation is
accomplished by combining the activities of ChOx and Ce-NCP,
which leads to a cascade reaction. Specifically, this cascade
reaction begins with the conversion of Chol by ChOx to cholest-
4-en-3-one and H2O2, and followed by the oxidation of TMB by
Ce-NCP to generate oxTMB with the assistant of the produced
H2O2 (Fig. 2b). On the other hand, the occurrence of this cascade
•
Fenton behavior in decomposing H2O2 to OH [12]. By further
investigating steady-state kinetics (Fig. S6 and Table S1), it was
found that compared with HRP, the Ce-NCP has a higher affinity
and a faster reaction rate to the substrates (H2O2 and TMB),
demonstrating its excellent catalytic performances.
reaction also reflects that Ce-NCP as
a compartment is
permeable to Chol. This is further demonstrated by the enhanced
oxTMB absorbance at high concentration of Chol (Fig. S12).
Taken together, ChOx@Ce-NCP has been successfully
fabricated and it is capable of driving one-pot cascade reaction.
Figure 1. (a) Colorimetric assay of coomassie brilliant blue G‐250 with Ce-NCP
and ChOx@Ce-NCP. (b) Relative absorbance of coomassie brilliant blue G‐250
in the presence of SDS-treated ChOx@Ce-NCP and the mixture of ChOx and
Ce-NCP.
The self-assembly of Ce3+, ATP and ChOx leads to the
formation of ChOx@Ce-NCP. By staining with coomassie brilliant
blue G‐250 (Fig. 1a), we can see that ChOx@Ce-NCP exhibits a
bright blue with the maximum absorbance at 595 nm, whereas the
absorption spectrum and color of Ce-NCP are the same as that of
the dye control. This suggests that ChOx has been presented,
which is confirmed by the observation of characteristic amide I
band (1645 cm−1) and II band (1510 cm−1) of protein in FTIR
spectrum (Fig. S7). On this basis, the immobilization mode of
ChOx in Ce-NCP was investigated by treating ChOx@Ce-NCP
with excess sodium dodecylsulfate (SDS). Under this condition,
any surface-absorbed proteins will be removed. However, we
found from Fig. 1b that after washing thoroughly, the protein
colorimetric assay was not affected by the SDS treatment,
demonstrating that ChOx was indeed entrapped inside the Ce-
NCP rather than adsorbed on its surface. With the calibration
curve in Fig. S8, the encapsulation efficiency of ChOx in Ce-NCP
was determined as 79.1 %, which is much higher than that of
conventional hosts such as porous silica (15 %) [13]. Nevertheless,
the ChOx@Ce-NCP still remained the same morphology (Fig.
S9) and structure (Fig. S10) as pure Ce-NCP, suggesting that the
structure of Ce-NCP is free from ChOx. This is verified by the
negligible changes of ChOx@Ce-NCP in the average
hydrodynamic diameter (Fig. S11). The unchanged structure of
ChOx@Ce-NCP can be attributed to the adaptive inclusion
property of Ce-NCP as a compartment, which follows the shape
of ChOx to flexibly rearrange its structures during self-assembly
Figure 2. (a) Absorption spectra and photography of TMB solutions with ChOx,
Ce-NCP and ChOx@Ce-NCP after adding Chol. (b) Schematic illustration of
Chol-triggered TMB oxidation by ChOx@Ce-NCP. (c) Time-dependent
absorbance and color changes of oxTMB catalyzed by ChOx@Ce-NCP and the
mixture system of free ChOx and Ce-NCP. (d) Relative activity of ChOx@Ce-
NCP and ChOx&HRP@ZIF-8 with molar ratios of ChOx to Ce-NCP (or HRP)
from 1:1 to 50:1.
The catalytic performances of ChOx@Ce-NCP were then
evaluated by monioring oxTMB absorbance at different intervals.
Fig. 2c shows that upon reaction time increasing, ChOx@Ce-
NCP can lead to an intensified oxTMB absorbance, which is same
as that of the mixture system of free ChOx and Ce-NCP. The
same trends indicate that these two cascade systems share a
same catalytic pathway. However, ChOx@Ce-NCP brings a
higher oxTMB absorbance at all stages, and an obvious 3-min lag
phase of oxTMB absorbance increase was found in the mixture
system. This reflects that ChOx@Ce-NCP can induce a faster
TMB conversion, which is consistent with its higher catalytic
activity (Fig. S13). Since identical reaction conditions are used in
these two cascade systems, the superior activity of ChOx@Ce-
NCP is attributed to the close proximity of ChOx to Ce-NCP within
a confined space. In this case, the intermediate H2O2 is able to be
processes [10a]
.
Since ChOx can catalyze cholesterol (Chol) to produce H2O2, a
cascade reaction might be initiated by Chol with the formation of
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generated in site and presented with a high local concentration in
the confined space of Ce-NCP. As a result, the produced H2O2
will be allowed to effectively transfer from ChOx to Ce-NCP
without leaving the confined space, which minimizes the diffusion
losses of H2O2 during transportation, and consequently leading to
an enhanced cascade activity in the ChOx@Ce-NCP.
ChOx@Ce-NCP can be attributed to confinement effect of Ce-
NCP as a compartment, which reduces the ability of ChOx to
change its structural configuration. Next, the biological stability of
ChOx@Ce-NCP was examined in the presence of trypsin. As
shown in Fig. S16c, after exposure to excess trypsin for 24 h,
ChOx@Ce-NCP can retain 95 % of initial activity, while almost no
activity was measured in the mixture system of free ChOx and Ce-
NCP. This suggests that Ce-NCP can protect ChOx from the
degradation of trypsin, possibly because of the large size of
trypsin that limits its diffusion across Ce-NCP. Moreover,
compared with the mixture system of free ChOx and Ce-NCP,
Because high local concentration of intermediates can often
cause a substrate inhibition effect to give a low cascade activity
in the co-encapsulated cascade systems, the effect of H2O2 on
ChOx@Ce-NCP was investigated. To this end, we conducted a
series of Chol conversion experiments that catalyzed by
ChOx@Ce-NCP with different molar ratios of ChOx and Ce-NCP,
as local H2O2 concentrations were determined by ChOx amounts
[6a]. From Fig. 2d, it can be seen that upon the increase of ChOx
amounts, the TMB oxidation reactions almost no suppressed,
suggesting that high local concentration of H2O2 has a negligible
inhibition effect to the cascade activity of ChOx@Ce-NCP. By
contrast, under identical conditions, the cascade activity of the co-
encapsulated ChOx and HRP in ZIF-8 (ChOx&HRP@ZIF-8) was
significantly inhibited, yielding a much lower oxTMB absorbance.
Apparently, the substrate inhibition effect was not occurred in
ChOx@Ce-NCP, which may be ascribed to the excellent stability
of Ce-NCP at high concentration of H2O2 (Fig. S14). Therefore,
the dual functions of Ce-NCP (as enzyme host and artificial
enzyme) act synergistically to drive cascade reaction, leading to
a highly efficient conversion of Chol. In addition, the successful
fabrications of ChOx@Ce-NCP with different molar ratios of ChOx
and Ce-NCP also substantially implies that controllable mass ratio
of building blocks is able to be achieved in this catalyst system.
The steady-state kinetic experiments were performed to further
investigate the catalytic property of ChOx@Ce-NCP. Fig. S15
shows that Chol oxidation catalyzed by ChOx@Ce-NCP follows
typical Michaelis-Menten behavior. With the Lineweaver-Burk
plots, the maximum initial velocity (Vmax) of ChOx@Ce-NCP was
calculated to be 4.398 × 10−8 M s−1, which is 2.4-fold higher than
the mixture system of free ChOx and Ce-NCP (Table S2). This
enhancement is along with an increase in turnover number (Kcat),
demonstrating the higher catalytic activity of ChOx@Ce-NCP.
However, the affinity of ChOx@Ce-NCP to substrate (Chol) is
slightly lower than the mixture system of free ChOx and Ce-NCP,
as reflected by its relatively higher Km value. Moreover, compared
with the mixture system of free ChOx and Ce-NCP, the similar
Kcat/Km value of ChOx@Ce-NCP indicates that ChOx is intact
without any activity loss during the integration of ChOx with Ce-
NCP. This is mainly benefited from the adaptive inculsion property
of Ce-NCP, except for the contributions from mild sysnthesis
condition and good biocompatibilty of building blocks.
ChOx@Ce-NCP has
a superior storage stability at room
temperature (Fig. S16d). By conducting successive reaction
batches, it was also found that after 6 cycles, nearly 95 % of initial
activity still can be retained in ChOx@Ce-NCP (Fig. S16e). This
reflects the reusability of ChOx@Ce-NCP, which is benefit from
the negligible leakage of ChOx from Ce-NCP (Fig. S16f).
Figure 3. (a) Emission spectra of ChOx@Ce-NCP and ChOx@Ce-NCP:Tb
under normal fluorescent model and time-resolved model. Inset is the
photography of ChOx@Ce-NCP and ChOx@Ce-NCP:Tb under UV lamp. (b)
Emission spectra of ChOx@Ce-NCP:Tb in the presence of Chol. (c) Schematic
illustration of the Chol-initiated luminescent switching behavior of ChOx@Ce-
NCP:Tb.
Inspired by the fact that Ce3+ can transfer its absorbed energy
to sensitize Tb3+ [14], a luminescent artificial MEC was further
fabricated by doping Tb3+ in ChOx@Ce-NCP, which generates
ChOx@Ce-NCP:Tb. The successful doping of Tb3+ was
confirmed by the increased Zeta potential (Fig. S17) and
measured Tb element (Fig. S18). Nonetheless, the presence of
Tb3+ has no influence on the morphology of ChOx@Ce-NCP (Fig.
S19). Upon the excitation at 290 nm, ChOx@Ce-NCP:Tb emits a
green fluorescence and accompanied by the record of five
obvious emission peaks (red line in Fig. 3a). The peak at 375 nm
is consistent to the maximum emission of ChOx@Ce-NCP, while
the peaks at 490, 545, 584, and 620 nm correspond to the 5D4 to
7Fj (j = 3 - 6) electronic transitions of Tb3+. Since no fluorescence
was yielded from ChOx@Tb-NCP, which reflects that both ATP
and ChOx cannot sensitize Tb3+, the origination of Tb3+ emission
in ChOx@Ce-NCP:Tb is ascribed to the sensitization effect of
Ce3+ through an energy transfer process.
In addition to enhanced catalytic performances, ChOx@Ce-
NCP also displays outstanding stability under a wider range of
conditions. From Fig. S16a, we can see that after treating at 60
oC for 60 min, more than 70 % of initial activity was maintained in
ChOx@Ce-NCP, whereas the mixture system of free ChOx and
Ce-NCP was completely inactive. Similarly, in the presence of
urea, a well-known denaturant, a superior activity was also
observed in ChOx@Ce-NCP, especially at a high concentration
of urea (Fig. S16b). The results indicate that the stability of
ChOx@Ce-NCP against high temperature and urea has been
greatly improved. Since the catalytic activity of Ce-NCP was not
affected by high temperature and urea, the enhanced stability of
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Upon the occurrence of energy transfer from Ce3+ to Tb3+, a
time-resolved fluorescence can be measured in ChOx@Ce-
NCP:Tb. Fig. 3a shows that under a time-resolved fluorescent
mode, ChOx@Ce-NCP:Tb exhibits only Tb3+ emission (blue line),
but the peak at 365 nm (corresponding to Ce3+) does not be
recorded. Meanwhile, it was found that the Tb3+ emission is much
stronger than that in the case of normal fluorescent mode. This
indicates that the background fluorescence of Ce3+ in ChOx@Ce-
NCP:Tb can be eliminated under time-resolved fluorescent mode,
resulting in a high signal-to-noise ratio of Tb3+. Fig. S20 depicts
that to obtain best emission performances, the most suitable ratio
of Ce3+ to Tb3+ in ChOx@Ce-NCP:Tb was at 4:1. On this basis,
we discussed the role of ChOx in the emission behaviors of
ChOx@Ce-NCP:Tb. From Fig. S21, it can be seen that under
same conditions, ChOx@Ce-NCP:Tb has a stronger emission
intensity and a longer emission lifetime as compared to the
mixture system of free ChOx and Ce-NCP:Tb, which is consistent
with its higher quantum yields (38.87 % vs 19.67 %). This reflects
that the encapsulation of ChOx in Ce-NCP:Tb can greatly
enhance the emission performances of Tb3+, which may due to
the elimination of the quenching effect of water molecule on Tb3+
fluorescent assay of Chol. Therefore, this work provides a new
way to fabricate artificial MEC and which may find applications in
biomedical diagnostics and analyses.
Acknowledgements
We gratefully acknowledge the financial support from the Natural
Science Foundation of China (21765010 and 21305054).
Keywords: Artificial multi-enzyme complex • Cascade reaction •
Catalytic compartment • Nanoscale coordination polymer • Time-
resolved fluorescent assay
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demonstrated to drive
a cascade amplified time-resolved
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Entry for the Table of Contents
Layout 1:
COMMUNICATION
The doping of Tb(III) in ChOx@Ce-
NCP leads to a luminescent artificial
multi-enzyme complex (ChOx@Ce-
NCP:Tb), which combines the merits
of cascade catalysis of artificial multi-
enzyme complex and unique emission
behaviour of Tb(III) and is capable of
driving a cascade amplified time-
resolved fluorescent assay within a
confined space, showing high
Jie Gao, Caihong Wang, Jinhong Wang,
Hongliang Tan*
Page No. – Page No.
Cascade amplified time-resolved
fluorescent assay driven by enzyme-
integrated catalytic compartment as
an artificial multi-enzyme complex
sensitivity and specificity toward
cholesterol.
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