Monitoring of Heparin Activity in Live Rats Using Metal-Organic Framework Nanosheets as Peroxidase Mimics

Article abstract of DOI:10.1021/acs.analchem.7b02895

Metal-organic framework (MOF) nanosheets are a class of two-dimensional (2D) porous and crystalline materials that hold promise for catalysis and biodetection. Although 2D MOF nanosheets have been utilized for in vitro assays, ways of engineering them into diagnostic tools for live animals are much less explored. In this work, a series of MOF nanosheets are successfully engineered into a highly sensitive and selective diagnostic platform for in vivo monitoring of heparin (Hep) activity. The iron-porphyrin derivative is selected as a ligand to synthesize a series of archetypical MOF nanosheets with intrinsic heme-like catalytic sites, mimicking peroxidase. Hep-specific AG73 peptides as recognition motifs are physically adsorbed onto MOF nanosheets, blocking active sites from nonspecific substrate-catalyst interaction. Because of the highly specific interaction between Hep and AG73, the activity of AG73-MOF nanosheets is restored upon the binding of Hep, but not Hep analogues and other endogenous biomolecules. Furthermore, by taking advantages of biocompatibility and diagnostic property enabled by AG73-MOF nanosheets, the elimination process of Hep in live rats is quantitatively monitored by coupling with microdialysis technology. This work expands the biomedical applications of 2D MOF nanomaterials and provides access to a promising in vivo diagnostic platform.

Full text of DOI:10.1021/acs.analchem.7b02895

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Article
Monitoring of Heparin Activity in Live Rats Using Metal-
Organic Framework Nanosheets as Peroxidase Mimics
Hanjun Cheng, Yufeng Liu, Yihui Hu, Yubin Ding, Shichao Lin, Wen Cao, Qian Wang,
Jiangjiexing Wu, Faheem Muhammad, Xiaozhi Zhao, Dan Zhao, Zhe Li, Hang Xing, and Hui Wei
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02895 • Publication Date (Web): 10 Oct 2017
Downloaded from http://pubs.acs.org on October 11, 2017
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Analytical Chemistry
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Monitoring of Heparin Activity in Live Rats Using Metal-Organic
Framework Nanosheets as Peroxidase Mimics
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Hanjun Cheng , Yufeng Liu , Yihui Hu , Yubin Ding , Shichao Lin , Wen Cao , Qian Wang , Jiangjiexꢀ
†
†
‡
§
†
*§
*
†
ing Wu , Faheem Muhammad , Xiaozhi Zhao , Dan Zhao , Zhe Li , Hang Xing, and Hui Wei
†
Department of Biomedical Engineering, College of Engineering and Applied Sciences, Collaborative Innovation Center of
Chemistry for Life Sciences, Nanjing National Laboratory of Microstructures, Nanjing University, Nanjing, Jiangsu 210093,
China.
‡
Department of Urology, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, Nanꢀ
jing, Jiangsu 210008, China.
§
Institute of Chemical Biology and Nanomedicine, College of Chemistry and Chemical Engineering, Hunan University,
Changsha, Hunan, 410082, China.
*
Eꢀmail: weihui@nju.edu.cn. Phone: +86ꢀ25ꢀ83593272. Fax: +86ꢀ25ꢀ83594648.
*
Eꢀmail: hangxing@hnu.edu.cn.
ABSTRACT: Metalꢀorganic framework (MOF) nanosheets are a class of twoꢀdimensional (2D) porous and crystalline materials
that hold promise for catalysis and biodetection. Although 2D MOF nanosheets have been utilized for in vitro assays, ways of engiꢀ
neering them into diagnostic tools for live animals are much less explored. In this work, a series of MOF nanosheets are successfulꢀ
ly engineered into a highly sensitive and selective diagnostic platform for in vivo monitoring of heparin (Hep) activity. The ironꢀ
porphyrin derivative is selected as ligand to synthesize a series of archetypical MOF nanosheets with intrinsic hemeꢀlike catalytic
sites, mimicking peroxidase. Hepꢀspecific AG73 peptides as recognition motifs are physically adsorbed onto MOF nanosheets,
blocking active sites from nonꢀspecific substrateꢀcatalyst interaction. Due to the highly specific interaction between Hep and AG73,
the activity of AG73ꢀMOF nanosheets is restored upon the binding of Hep, but not Hep analogues and other endogenous biomoleꢀ
cules. Furthermore, by taking advantages of biocompatibility and diagnostic property enabled by AG73ꢀMOF nanosheets, the elimꢀ
ination process of Hep in live rats is quantitatively monitored by coupling with microdialysis technology. This work expands the
biomedical applications of 2D MOF nanomaterials and provides access to a promising in vivo diagnostic platform.
Nanomaterials with enzymeꢀlike catalytic activities, known
as “nanozymes”, have received growing interest in the past
decades. Indeed, some intrinsic limitations arising from natuꢀ
ic proteases for protein hydrolysis and to monitor glucose levꢀ
27,28,36ꢀ38
el in living brains as peroxidases mimics.
However, the
1
potential drawback of bulk MOFsꢀbased nanozymes is that
only a small fraction of active sites is exposed on surface
while the majority of them are hidden within the framework.
Thus the catalytic activities are significantly impaired due to
the large diffusion barrier. In addition, the small surfaceꢀtoꢀ
volume ratio of bulk MOF nanozymes limits the number of
potential binding sites for interfacing biorecognition, which
further limits their biodiagnostic applications. To improve the
catalytic and biorecognition properties of MOF nanozymes, an
effective strategy is to engineer bulk MOF structure into ulꢀ
ral enzymes, such as low stability, high cost, and sensitivity to
harsh environments, all can be overcome to some extent
through the usage of nanozymes as a mimic. Up to now,
nanozymes from different nanomaterials have successfully
mimicked a series of natural enzymes, such as peroxidase,
oxidase, catalase, superoxide dismutase, nuclease, and phosꢀ
1
ꢀ15,
phatase,
which has enabled a variety of applications in
16ꢀ30
research and medicine.
Particularly, some intriguing appliꢀ
cations, such as rapid Ebola diagnosis and tumor imꢀ
munostaining, have been achieved by peroxidaseꢀmimicking
31,32,34
trathin twoꢀdimensional (2D) MOF nanosheet.
The 2D
3
,24
nanomaterials.
MOF nanozymes surpass their bulk analogues for (i) highly
exposed surface area with more accessible active sites for enꢀ
zymatic catalysis and (ii) high density of binding sites for inꢀ
teracting with targets of interest. Though 2D MOF nanosheets
have been reported as bioassays, ways of incorporating recogꢀ
nition motifs and further engineering those for in vivo biodiꢀ
agnostic application are still challenging.
Among all materials that mimic enzyme activities, metalꢀ
organic frameworks (MOFs), a class of crystalline and porous
materials formed by metal nodes and polydentate ligands,
3
1ꢀ35
have emerged as promising biomimetic catalysts.
Some
key features, such as multiple catalytic sites from either metal
nodes and/or ligands with active sites, tailorable structures,
high robustness to environment and convenient recyclability,
Herein, we describe the development of an in vivo bioassay
using 2D MOF nanosheets as peroxidase mimics. A series of
2D MOFs were synthesized from binuclear paddleꢀwheel metꢀ
have extended the application region of MOFs over other maꢀ
31ꢀ35
terials.
Indeed, MOFs have been reported recently to mimꢀ
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al clusters and metallated tetrakis(4ꢀcarboxyphenyl)porphyrin Then, through a midline cervical incision, both common carotꢀ
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(TCPP) ligands to mimic peroxidase. The prepared 2D MOF
nanozymes possess enhanced peroxidaseꢀmimicking activities
than their 3D bulk analogues. Using TCPP metallated with
different metal ions, we identified 2D MOF architecture conꢀ
sisting of Feꢀbound TCPP (TCPP(Fe)) ligands exhibit the
highest activity, demonstrating the dominant role of the hemeꢀ
like ligands in determining the activities of nanozymes. As
proofꢀofꢀconcept of bioassay, antiꢀheparin (Hep) peptides
AG73 were physically adsorbed onto MOF nanosheets, moduꢀ
lating the enzymatic activity by interacting with peptides and
Hep molecules. Furthermore, AG73ꢀ2D MOF as a sensitive
and selective bioassay was demonstrated to monitor Hep elimꢀ
ination process in live rats.
id arteries were exposed and isolated from surrounding conꢀ
nective tissue. A linear microdialysis probe (CMA) was then
carefully embedded in one carotid artery. Note, particular care
was needed to avoid damaging the vagus and the sympathetic
nerves close by. The linear microdialysis probe was perfused
with Ringer’s solution at 1 ꢁL/min. The perfusion was run for
at least 60 min to achieve equilibration before collection of
microdialysis sample. After equilibration, the rat was adminisꢀ
trated with Hep by intraperitoneal injection of 2 mL Hep saꢀ
line solution (100 ꢁg/mL). Throughout the surgery, the body
temperature of the animals was maintained at 37°C using a
heating pad.
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Instrumentation. Powder Xꢀray diffraction (PXRD) data
were collected on an ARL SCINTAG X’TRA diffractometer
using Cu Kα radiation (Thermo). Transmission electron miꢀ
croscopy (TEM) was performed on a Tecnai F20 transmission
electron microscope (FEI) at an acceleration voltage of 200
kV. The hydrodynamic size of 2D ZnꢀTCPP(Fe) nanosheet
was measured on a Nanosizer ZS90 (Malvern). UVꢀvisible
absorption spectra were collected on a UVꢀvisible spectrophoꢀ
tometer with a 1ꢀcm quartz cell (Beijing Purkinje General
Instrument Co. Ltd., China). Fluorescent spectra were obꢀ
tained on a Hitachi Fꢀ4600 fluorescent spectrometer (Japan).
EXPERIMENTAL SECTION
In vitro measurement of Hep. To develop the 2D MOFꢀ
based bioassay for measurement of Hep, AG73 peptide (10
ꢁg/mL) and 2D ZnꢀTCPP(Fe) nanosheets (5 ꢁg/mL) were preꢀ
incubated in 0.10 M acetate buffer (pH 5.0) for 5 min to form
peptide/ZnꢀTCPP(Fe) nanocomposites. Then, different conꢀ
centrations of Hep in Ringer’s solution were added into the
mixture and the resulting reaction solution was incubated at
room temperature for another 40 min to allow complete bindꢀ
ing between AG73 and Hep. Finally, the reacted solution was
mixed with H O and chromogenic substrate TMB or Ampliꢀ
RESULTS AND DISCUSSION
2
2
flu Red to allow UVꢀvisible spectroscopic measurements. By
using TMB as the reporting molecule, the obtained reaction
mixtures containing 1 mM H O and 500 ꢁM TMB were alꢀ
Synthesis of 2D MOF Nanosheets and Evaluation of
Their Peroxidase-like Activities. To design a 2D MOF
nanosheet mimicking peroxidase, both the MOF structure as a
whole and the active sites within the network need to be conꢀ
sidered. A type of 2D MOF structures consisting of binuclear
2
2
lowed to UVꢀvisible spectroscopic measurements by continuꢀ
ously monitoring the absorption changes at 652 nm. On the
other hand, by using Ampliflu Red as the reporting molecule,
the obtained reaction mixtures containing 1 mM H O and 400
2+
2+
2+
paddleꢀwheel inorganic units (e.g., Zn , Co , and Cu ) and
hemeꢀlike metalloporphyrin polydentate organic ligands (e.g.,
2
2
3
1,32
ꢁM Ampliflu Red were allowed to fluorescent spectroscopic
TCPP(Fe)) was selected as model system (Figure 1A).
In
measurements by continuously monitoring the fluorescence
emission spectra at 585 nm with the excitation wavelength at
this type of structure, both organic and inorganic building
blocks feature planar 4ꢀconnectivity, ensuring the growth of
the coordination network into a 2D plane rather than a 3D
extended structure. In addition, as heme is a cofactor for natuꢀ
ral peroxidases, it is expected that the incorporated metalꢀ
loporphyrins would be able to render the MOF structure with
5
60 nm.
In vivo measurement of Hep in live rats. The animal studꢀ
ies were approved by the Committee for Experimental Aniꢀ
mals Welfare and Ethics of Nanjing Drum Tower Hospital, the
Affiliated Hospital of Nanjing University Medical School.
Adult male Sprague−Dawley rats (250ꢀ300 g) were purchased
from Jiesijie Laboratory Animal Co. (Shanghai, China). Hep
saline solution (0.9%) of 2 mL (100 ꢁg/mL) was injected inꢀ
traperitoneally. After dosing for 20 min, 1 mL of blood was
first taken from the abdominal aorta of the rats and was then
purified by perfusing the fluids through a microdialysis probe
39
required peroxidase mimicking activities. The 2D MOF
2
+
2+
nanozymes consisting of divalent metal ions Zn , Co or
2+
Cu and ligand TCPP(Fe) (termed as 2D ZnꢀTCPP(Fe), 2D
CoꢀTCPP(Fe), and 2D CuꢀTCPP(Fe)) were prepared via a
surfactantꢀassisted strategy, where PVP was used as surfactant
to confine the growth of MOF crystal into two dimensions.
Transmission electron microscopy (TEM) was used to visualꢀ
ize the structure of prepared MOFs. As shown in Figure 1B
and S1, all three 2D MOFs exhibited wellꢀdefined ultrathin
sheetꢀlike structures. The 2D sheet structure of 2D Znꢀ
TCPP(Fe) was further studied by atomic force microscopy
(CMA, 4 ꢁm length) at 1.0 ꢁL/min.
For the measurement of Hep in the blood of live rats, 10 ꢁL
of AG73 (100 ꢁg/mL) and 0.8 ꢁL ZnꢀTCPP(Fe) (612 ꢁg/mL)
were added into 60 ꢁL 0.10 M acetate buffer (pH 5.0) and
incubated for 5 min to prepare a fresh probe solution. Then, 20
(AFM) (Figure 1C). The thickness of 2D ZnꢀTCPP(Fe) was
estimated ca. 1 ꢀ 1.5 nm (Figure 1D), indicating only a few
molecular layers were stacked. The crystalline feature of these
ꢁ
L of the sampled blood microdialysate was added into the
probe solution and the resultant mixture was incubated for
another 40 min. Finally, 10 ꢁL of Ampliflu Red (4 mM) and 1
2
D MOF nanozymes was characterized by powder Xꢀray difꢀ
fraction (PXRD). As shown in Figure 1E, all MOFs displayed
three feature peaks at 7.6°, 8.8°, and 17°, which were indexed
as (110), (002), and (004), respectively, suggesting the assemꢀ
ꢁ
L H O (100 mM) were added into the reaction mixture and
2 2
the solution was immediately processed for fluorescent specꢀ
troscopic measurements by continuously monitoring the fluoꢀ
rescence emission at 585 nm with the excitation wavelength at
31,32
bled (4, 4) 2D network.
5
60 nm.
The peroxidaseꢀmimicking activities of the prepared 2D
MOF nanozymes were evaluated through the oxidation of
In vivo monitoring Hep elimination process. The rats
peroxidase
chromogenic
substrate
3,3’,5,5’ꢀ
were firstly anesthetized with chloral hydrate (345 mg/kg, ip).
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Analytical Chemistry
kinetics of enzymatic reaction among three 2D MOF
tetramethylbenzidine (TMB) with H O and characterized by
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UVꢀvisible spectroscopy (UVꢀvis). As shown in Figure 1F, the
introduction of the 2D ZnꢀTCPP(Fe) into 0.10 M acetate buffꢀ
er (pH 5.0) containing both H O and TMB resulted in an imꢀ
nanosheets (ZnꢀTCPP(Fe), CoꢀTCPP(Fe), and CuꢀTCPP(Fe))
were further studied by monitoring the characteristic absorpꢀ
tion peak of TMBox centered at 652 nm. All three nanozymes
with different metal nodes exhibited comparable kinetic
curves in 300 s with no significant differences of the catalytic
activities were observed, indicating metal nodes may have no
correlation with the enzymatic property (Figure S2).
2
2
mediate color change from colorless to blue with a characterisꢀ
tic UVꢀvis absorption peak of oxidized TMB (TMB ) cenꢀ
ox
tered at 652 nm. In contrast, the combination of any of the two
components did not generate color change, demonstrating the
peroxidaseꢀmimicking activity of the 2D MOF nanozyme. The
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Figure 1. (A) Scheme showing the surfactantꢀassisted bottomꢀup synthesis of 2D MOF nanosheets. (B) TEM image of 2D Znꢀ
TCPP(Fe) nanosheet. (C) AFM image of 2D ZnꢀTCPP(Fe) nanosheet and (D) the corresponding crossꢀsectional analysis of three
pieces of nanosheets. The heights of all three pieces were estimated ca. 1 – 1.5 nm. (E) PXRD patterns of 2D CoꢀTCPP(Fe) (black),
ZnꢀTCPP(Fe) (dark grey), and CuꢀTCPP(Fe) (grey) nanosheets. (F) UVꢀvisible absorption spectra of six sample solutions in 0.10 M
acetate buffer (pH 5.0) containing (1) 2D ZnꢀTCPP(Fe) + TMB + H O , (2) 2D ZnꢀTCPP(Fe) + TMB, (3) 2D ZnꢀTCPP(Fe) +
2
2
H O , (4) H O + TMB, (5) TMB, (6) H O . Inset: corresponding photograph image of sample solutions in test tubes. (G) Kinetic
2
2
2
2
2
2
curves plotting the timeꢀdependent UVꢀvis absorbance at 652 nm of reactions catalyzed by 2D and 3D bulk ZnꢀTCPP(Fe) MOFs,
showing the different catalytic properties.
To systematically identify whether the catalytic activities of
2D MOF nanozymes are stemmed from their chelated metal
ions or from their hemeꢀlike metalloporphyrin ligands, experꢀ
iments studying the effects of metal nodes in MOF structure
and porphyrinꢀcoordinated metal ions were carried out. To
study the effect of metal nodes and minimize the influence of
porphyrin center, three 2D MOF nanosheets (Zn-TCCP, Co-
TCCP, and Cu-TCCP) were synthesized using TCPP ligand
with empty porphyrin core. Ultrathin 2D sheet structures were
observed for all three MOFs under TEM images (Figure S3).
Peroxidaseꢀmimicking activities of all three MOFs were moniꢀ
tored using the same method described above. As shown in
Figure S4, all three MOFs with empty porphyrin core exhibitꢀ
ed extremely low catalytic activities, invalidating the possibilꢀ
ity of an active metal nodes in MOF (Figure S4). To study the
effect of porphyrinꢀcoordinated metal ions, TCPP(Zn),
TCPP(Co) and TCPP(Mn) in addition to TCPP(Fe) were used
2+
to prepare 2D MOF structures with Zn cluster as the metal
nodes. Interestingly, ZnꢀTCPP(Zn) and ZnꢀTCPP(Co) exhibitꢀ
ed similar sheetꢀlike 2D structures to ZnꢀTCPP(Fe), while the
obtained ZnꢀTCPP(Mn) tended to form nanobelt morphology
(
Figure S5). The enzymatic activity studies clearly showed
that none of the MOF structures have comparable peroxidaseꢀ
mimicking activities to ZnꢀTCPP(Fe), the activity of which is
ca. 7ꢀ20 times higher than MOFs with nonꢀFe porphyrin ligꢀ
ands (Figure S6). This is expected because Fe center serves as
the active site in natural peroxidase. Taken together, these
results support the conclusion that the TCPP ligand played a
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dominant role in determining the peroxidaseꢀmimicking activiꢀ
ties of 2D MOF nanozymes while the connecting metal nodes
mainly serve as structural building blocks, i.e., the porphyrin
centerꢀlike TCPP ligands acted as the active sites in 2D MOF
nanosheets. For all the MOF structures studied, the ones conꢀ
taining TCPP(Fe) exhibited the highest catalytic activities. As
at right dosage but induces severe side effects if overdose ocꢀ
40ꢀ42
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curs.
And thus there is a need for realꢀtime monitoring the
43ꢀ46
elimination process of Hep.
To achieve targetꢀspecific activation of the enzymatic propꢀ
erty on MOF, a Hepꢀspecific binding peptide AG73 with the
sequence of RKRLQVQLSIRT and isoelectric point (pI) of
12.4 was immobilized onto 2D ZnꢀTCPP(Fe) surface through
2
D ZnꢀTCPP(Fe) was among the best peroxidase mimics disꢀ
44,47
cussed above, it was used as a model material for further studꢀ
ies.
physical adsorption.
To confirm the immobilization of
peptides on the 2D MOF surface, we probed the surface
charge of 2D ZnꢀTCPP(Fe) nanosheets with the addition of
AG73. As shown in Figure S15, the ζꢀpotential of unmodified
2D ZnꢀTCPP(Fe) nanosheets was ꢀ15.3 mV, mainly attributed
to the uncoordinated carboxyl groups on surface. The addition
of AG73 led to an increment of the resultant ζꢀpotential. Since
the net charge of the peptide is estimated to be positive under
the experimental condition, we attribute the ζꢀpotential change
of MOF nanosheets to the electrostatic binding of AG73 to 2D
ZnꢀTCPP(Fe) nanosheets. In addition, the ζꢀpotential of the
AG73ꢀ2D ZnꢀTCPP(Fe) complex was ca. +15.1 mV after addꢀ
ing 10 ꢁg/mL AG73 and reached a plateau with higher peptide
concentration, suggesting that the adsorption of AG73 onto 2D
ZnꢀTCPP(Fe) nanosheets was saturated.
To study the advantages of 2D MOF nanosheet, a 3D bulk
2
+
analogue was prepared from Zn and TCPP(Fe) in the abꢀ
sence of PVP. As shown in Figure S7A, the 3D ZnꢀTCPP(Fe)
exhibited microꢀsized irregular morphology. The PXRD difꢀ
fraction patterns of 2D and 3D ZnꢀTCPP(Fe) matched well
with each other, indicating the same crystalline structure (Figꢀ
ure S7B). The peroxidaseꢀmimicking activity of 3D Znꢀ
TCPP(Fe) was investigated and compared with that of 2D Znꢀ
TCPP(Fe). As shown in Figure 1G and S8, a faster kinetic rate
and a two times higher catalytic activity of 2D ZnꢀTCPP(Fe)
than these of 3D ZnꢀTCPP(Fe) were observed. The results
indicate that by engineering 3D MOFs into 2D ones, their
peroxidaseꢀmimicking activities can be significantly enhanced.
Such enhancement is possibly attributed to larger exposed
surface area with more accessible catalytic sites (i.e., the
TCPP(Fe)) and less diffusion barrier of 2D ZnꢀTCPP(Fe)
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To study the impact of AG73 modification on the peroxiꢀ
daseꢀlike activity of 2D ZnꢀTCPP(Fe) nanosheets, the kinetics
and activity profile of the enzymatic reaction were studied by
varying AG73 concentration and keeping MOF concentration
constant (Figure 2A). Ampliflu Red or TMB as a peroxidase
substrate was used as signal readout (Figure 2A and S16). As
shown in Figure 2B and Figure 2C, higher AG73 concentraꢀ
tion resulted in a slower catalytic rate and a lower peroxidaseꢀ
like activity, indicating the adsorption of AG73 on nanosheet
surface shields the catalytic sites. The AG73ꢀinduced inhibiꢀ
tion of peroxidaseꢀlike activity of MOF reached a plateau after
the addition of 10 ꢁg/mL AG73, which matches well with ζꢀ
potential measurements, confirming AG73 adsorption reached
equilibrium state (Figure 2C).
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nanosheets.
Tuning Peroxidase-like Activity of 2D Zn-TCCP(Fe) us-
ing Anti-Hep AG73 Peptide. After systematically studied the
structuralꢀactivity relationship of 2D MOF nanozymes, Znꢀ
TCPP(Fe) with the highest peroxidaseꢀmimicking activity was
chosen as model complex for bioassay development. To deꢀ
velop an enzymatic bioassay, the activity of enzyme should be
designed under quenching mode in the absence of targets and
restored in the presence of targets. As proofꢀofꢀconcept of
bioassay development, a negatively charged linear glycosaꢀ
minoglycan, Hep was chosen as the target of interest. As an
anticoagulant medication, Hep prevents deep vein thrombosis
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Figure 2. (A) Schematic illustration of AG73ꢀinhibited peroxidaseꢀlike activity of 2D ZnꢀTCPP(Fe) nanosheets with Ampliflu Red
as the redox substrate. (B) Kinetic curves plotting the timeꢀdependent fluorescence emission intensity at 585 nm (FI585) for Ampliꢀ
flu Red oxidation reactions catalyzed by ZnꢀTCPP(Fe) in different AG73 concentrations. The reactions were processed in 0.10 M
acetate buffer (pH 5.0) containing 400 ꢁM Ampliflu Red, 1 mM H O , and 5 ꢁg/mL 2D ZnꢀTCPP(Fe) nanosheets. (C) Normalized
catalytic activity of 2D ZnꢀTCPP(Fe) nanosheets in different AG73 concentrations. The catalytic activity decreases with the inꢀ
crease of peptide concentration. Inset: plot of normalized activity versus the logarithm of the AG73 concentration, showing a linear
relationship in low concentration range.
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To further understand the peptideꢀMOF interaction, activiꢀ
ties of free TCPP(Fe) ligands and bulk ZnꢀTCPP(Fe) MOFs
modulated by AG73 of different concentrations were studied
tivity of peptideꢀmodified MOF was quenched with minimum
fluorescence emission. After the addition of Hep, AG73 pepꢀ
tides on MOF surface were able to specifically recognize Hep,
destabilize the peptideꢀMOF interaction, and trigger the AG73
release from the surface of AG73ꢀ2D ZnꢀTCPP(Fe) complex,
resulting in the recovery of peroxidaseꢀlike activity and inꢀ
creased fluorescence signal. The recovered peroxidaseꢀlike
activity was measured by monitoring the timeꢀdependent fluoꢀ
rescence intensity of Resorufin (i.e., the oxidized product of
Ampliflu Red) at 585 nm, which is positively correlated to the
target Hep concentration (Figure 3B). As shown in Figure 3C,
a more quantitative analysis was carried out by plotting the
ratios of fluorescence intensity change ((F ꢀF )/(F ꢀF )) after
(
Figure S17). For free TCPP(Fe), the catalytic activity reꢀ
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mained almost unchanged in the presence of AG73 in the testꢀ
ed concentration range, suggesting the negligible peptideꢀ
ligand interaction. For bulk ZnꢀTCPP(Fe) MOF, the peroxiꢀ
daseꢀmimicking activity increased with the increase of AG73
concentrations, which can be attributed to peptideꢀpromoted
solubilization of MOF particles. Taken together, the data sugꢀ
gest the activity of 2D ZnꢀTCPP(Fe) nanosheets can be moduꢀ
lated by a Hepꢀspecific peptide but not the free Feꢀporphyrin
ligand or the bulk MOF particle, supporting our hypothesis of
using 2D ZnꢀTCPP(Fe) for bioassay development.
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0
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0
adding Hep versus the logarithm of Hep concentrations. A fit
of the data indicates a linear response curve ranging from 0.1
– 10 ꢁg/mL Hep with the slope of ca. 0.43 and a yꢀintercept
Detection of Hep with 2D Zn-TCPP(Fe) Nanozyme. Havꢀ
ing demonstrated the antiꢀHep peptide AG73ꢀmodified 2D Znꢀ
TCPP(Fe) nanosheets with quenched peroxidaseꢀlike activity,
we then explored their Hepꢀspecific activation and application
as competitive bioassay for Hep detection. Figure 3A shows
the Hep detection process using Ampliflu Red as the redox
substrate for reporting signal. In the absence of Hep, the acꢀ
2
of ca. 0.49 (R = 0.991). The detection limit was further deꢀ
termined to be ca. 15 ng/mL (S = 3σ). These data suggest that
the developed bioassay satisfies the requirements of monitorꢀ
ing Hep in clinical samples with concentrations ranging from
4
8,49
1
.1 to 6.5 ꢁg/mL during postoperative and longꢀterm care.
Figure 3. (A) Schematic illustration of AG73ꢀmodifed 2D MOFꢀbased bioassay for Hep detection. (B) Kinetic curves plotting the
timeꢀdependent fluorescence emission intensity at 585 nm for Ampliflu Red oxidation reactions catalyzed by AG73ꢀ2D Znꢀ
TCPP(Fe) in response to different Hep concentrations. The reactions were carried out in 0.10 M acetate buffer (pH 5.0) containing
4
00 ꢁM Ampliflu Red, 1 mM H O , 5 ꢁg/mL 2D ZnꢀTCPP(Fe) nanosheets, 10 ꢁg/mL AG73, and different concentrations of Hep.
2 2
(
C) Plot of fluorescence intensity change ratio (F ꢀF )/(F ꢀF ) versus the logarithm of the Hep concentration. F , the FI in the
2 0 1 0 0 585
presence of AG73; F , the FI values in the absence of Hep and AG73; F , the FI values in the presence of Hep and AG73. Error
1
585
2
585
bars indicate standard deviations of three independent measurements.
To allow monitoring Hep concentration in physiological
conditions, such as in live animals, the developed bioassay
must have high selectivity toward the target. To evaluate the
selectivity of the 2D MOFꢀbased bioassay, a variety of comꢀ
mon biological interfering species were tested for their effects
on nanozyme activation, including Hep analogues (chondrotin
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sulfate (CS) and hyaluronic acid (HA)), bioactive small moleꢀ also confirmed by ζꢀpotential measurements. Only Hep deꢀ
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cules (glucose, lactate, ascorbic acid (AA), uric acid (UA),
adenosine triphosphate (ATP), adenosine diphosphate (ADP),
adenosine monophosphate (AMP)), and biologically important
creased the ζꢀpotential of the AG73ꢀ2D ZnꢀTCPP(Fe) nanoꢀ
complex but not analogues, confirming the highly specific
recognition capacity of AG73 toward Hep. However, high
concentration of BSA (5 mg/mL) as representative of serum
protein was also observed to increase enzymatic activity, posꢀ
sibly due to the BSAꢀpromoted solubilization of 2D Znꢀ
TCPP(Fe) (Figure S18).
3
ꢀ
2ꢀ
ꢀ
+
2+
2+
anions and cations (PO , SO , NO , K , Mg , and Ca ).
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3
As shown in Figure 4A, none of them showed high fluoresꢀ
cence change ratio, indicating minimum interference for Hep
detection. The selectivity of developed bioassay for Hep was
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Figure 4. (A) Selectivity of the AG73ꢀ2D ZnꢀTCPP(Fe) bioassay toward Hep detection. Bars represent the fluorescence change
ratio after adding of Hep and other interfering molecules. Error bars indicate standard deviations of three independent measureꢀ
ments. (B) ζꢀpotentials of AG73ꢀ2D ZnꢀTCPP(Fe) nanocomplex measured in the absence or the presence of Hep, ChS or HA, reꢀ
spectively.
Figure 5. (A) Scheme showing the monitoring of Hep elimination process in live rats using 2D MOF nanozymes. (B) Kinetic
curves plotting the timeꢀdependent fluorescence intensity at 585 nm for three different samples mixing with AG73ꢀmodified MOFs
and redox substrate: (red curve) microdialyzed serum from Hepꢀtreated rats; (grey curve) microdialyzed serum from normal rats;
(black curve) negative control sample with no serum. (C) Dynamic changes of Hep concentrations in the artery of live rats over 4
hours following the administration of Hep. A fitting of the data indicates an exponential decay. Error bars indicate standard deviaꢀ
tions of three independent measurements.
To eliminate the potential interference from serum proteins
when applying the developed bioassay for Hep detection in
live rats, microdialysis technology was used to treat serum
sample before the measurement. We first tested the rat serum
samples spiked with different concentrations of Hep (i.e., 5,
mance of microdialysis. As shown in Figure S19 and Table
S1, after microdialysis, the 2D MOF bioassay responded norꢀ
mally to all three Hepꢀspiked serum samples with a consistent
ca. 65% detection recovery of spiked concentration. Next, to
further demonstrate the feasibility of using developed bioassay
for detecting Hep in live animals, Hep (100 ꢁg/mL, 2 mL) in
1
0, and 20 ꢁg/mL) after microdialysis to validate the perforꢀ
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0
.9% normal saline was dosed intraperitoneally to rats and the
AUTHOR INFORMATION
Corresponding Author
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blood samples were collected 20 min after dosage. The colꢀ
lected blood samples were microdialysed and then diluted 4ꢀ
fold with 0.10 M acetate buffer (pH 5.0) for measurements.
The average Hep concentration in live rats was determined to
be 4.47 ± 0.73 ꢁg/mL (Table S2), which is consistent with
*Eꢀmail: weihui@nju.edu.cn. Phone: +86ꢀ25ꢀ83593272. Fax:
+86ꢀ25ꢀ83594648. Web: weilab.nju.edu.cn.
*Eꢀmail: hangxing@hnu.edu.cn.
4
3
previous reports studying Hep metabolism, validating the use
of 2D MOF bioassay for in vivo diagnostic applications.
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manuꢀ
script.
The Hep elimination process in live rats was monitored by
using the AG73ꢀmodified MOF bioassay in combined with the
microdialysis probe. Figure 5A shows the design of the animal
experiment. A linear microdialysis probe was carefully emꢀ
bedded in the artery of live rats and the corresponding microꢀ
dialysates were continuously collected after the rats were inꢀ
traperitoneally administered with Hep (Figure 5A). A kinetic
study of the nanozyme catalytic reaction showed that microdiꢀ
alyzed serum from Hepꢀtreated rats significantly activated
AG73ꢀmodified MOF nanozymes while microdialyzed serum
from normal rats or negative control sample with no serum did
not (Figure 5B), suggesting a selective response of the bioasꢀ
say. The elimination process of the Hep was investigated by
plotting the time course of Hep concentrations in the artery of
live rats over 4 hours following administration (Figure 5C). As
shown in Figure 5C, after the rats were intraperitoneally inꢀ
jected with Hep for 0.5 hour, the Hep concentration in the
artery was determined to be 5.84 ± 1.04 ꢁg/mL and gradually
decreased overtime to 0.41 ± 0.17 ꢁg/mL after 4 hours due to
the elimination of Hep, which is possibly through depolymeriꢀ
zation into smaller fragments by the reticuloendothelial system
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ACKNOWLEDGMENT
This work was supported by National Natural Science Foundation
of China (21722503, 21405081, and 21405079), Natural Science
Foundation of Jiangsu Province (BK20160615), 973 Program
(2015CB659400), Shuangchuang Program of Jiangsu Province,
Open Funds of the State Key Laboratory of Analytical Chemistry
for Life Science (SKLACLS1704), Open Funds of the State Key
Laboratory of Coordination Chemistry (SKLCC1619), China
Postdoctoral Science Foundation (2016M590437), and Thousand
Talents Program for Young Researchers. The authors thank Dr.
Meiting Zhao for her assistance on 2D MOF synthesis.
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In summary, we developed a highly sensitive and selective
peptideꢀmodified 2D MOF nanosheet as diagnostic probe for
Hep. Hepꢀspecific peptide AG73 modification provided MOF
nanosheets with targetꢀresponsive catalytic activity, through
the stronger peptideꢀHep interactions than peptideꢀMOF interꢀ
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