Fabrication of highly active phosphatase-like fluorescent cerium-doped carbon dots for: In situ monitoring the hydrolysis of phosphate diesters

Article abstract of DOI:10.1039/d0ra07429b

Ce-Doped carbon dots (CeCDs) were fabricated via a one-step hydrothermal carbonization using Ce(NO3)3·6H2O and EDTA·2H2O as precursors, and various experimental techniques were employed to characterize the morphology, structure and composition of the as-o

Full text of DOI:10.1039/d0ra07429b

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Fabrication of highly active phosphatase-like
fluorescent cerium-doped carbon dots for in situ
monitoring the hydrolysis of phosphate diesters†
Cite this: RSC Adv., 2020, 10, 41551
Jinyan Du, * Shuangqing Qi, Juan Chen, Ying Yang, Tingting Fan, Ping Zhang,
Shujuan Zhuo and Changqing Zhu*
Ce-Doped carbon dots (CeCDs) were fabricated via a one-step hydrothermal carbonization using
3 3 2 2
Ce(NO ) $6H O and EDTA$2H O as precursors, and various experimental techniques were employed to
characterize the morphology, structure and composition of the as-obtained CeCDs. Using the disodium
salt of bis(4-nitrophenyl)phosphate (BNPP) as a DNA model substrate, mimetic phosphatase activity
toward phosphate ester hydrolysis cleavage was investigated. It was found that similar to the catalytic
character of the natural enzyme, CeCDs as a hydrolase mimic can exhibit a good catalytic activity for
promoting BNPP hydrolysis, in which metal Ce(III) acts as a center for binding and activation of the
metal-bound hydroxide complex as well as a source of nucleophilic metal hydroxides. Additionally,
based on the Inner Filter Effect (IFE), fluorescence spectra can be used to monitor the hydrolysis of
BNPP using CeCDs, which mimic phosphatase. Thus, the unique properties of the CeCD mimetic
phosphatases as well as the IFE sensing strategy would provide an ideal platform to monitor the catalytic
phosphate ester hydrolysis processes. Finally, to validate the availability of the established catalytic
systems, the CeCDs were further applied for degradation of organophosphorus pesticide chlorpyrifos.
The degradation efficiency was estimated to be a satisfactory value of 74.50%, exhibiting a potential
application prospect for treatment of the pollutants in the soil and water.
Received 29th August 2020
Accepted 7th November 2020
DOI: 10.1039/d0ra07429b
rsc.li/rsc-advances
these studies have shown that biological enzymes that catalyze
the hydrolysis of phosphates are always activated by one, two or
1
. Introduction
Phosphate esters are crucial for cell division and growth, more metal ions of the transition metal complexes. It was
information storage and utilization, energy transduction, believed that the metal ion can activate the phosphate group
cellular signaling communication, and other biological and a nucleophilic H
2
O molecule, and stabilize the penta-
1
processes in living systems. Hydrolytic cleavage of the P–O coordinated phosphorus transition state by cooperative effect
2
17
linkage of phosphate esters is the key step for these processes.
in metalloenzymes. These mimetic metallohydrolases not only
Generally, phosphate esters are highly stable with a half-life up have similar natural enzyme activities and high substrate
3
to thousands of years. Fortunately, natural biological enzymes specicities, but also effectively overcome the disadvantages of
can hydrolyze the phosphate ester bond rapidly with a high natural enzymes, such as low stabilities due to denaturation,
4
catalytic performance and a high specicity. However, poor sensitivity to the environment, difficult preparation and puri-
stability and easy inactivation limit their large-scale applica- cation, providing valuable information on the fundamental role
5
tions. Recently, fabrication of transition metal complexes as of metal ions in promoting the hydrolysis reactions of phos-
articial hydrolytic metalloenzymes for facilitating phosphate phate ester.
6
–9
diester hydrolysis has received considerable attention.
In particular, the signicant hydrolytic abilities of lantha-
II 10
II 11
II 12
III 13
II 14
II 15
18–21
Investigations on Zn , Ni , Cu , Fe , Co , Mg , and nide metal have caused a widespread concern.
In compar-
II
Mn (ref. 16) complexes as hydrolytic metalloenzymatic models ison to biologically relevant transition metals or alkaline earth
were successively reported. From a perspective of mechanism, Lewis acids, trivalent metal ions like lanthanum(III) exhibit high
catalytic activities toward P–O bond scission due to high Lewis
acidity, oxidation state and charge density, coordination
Anhui Key Laboratory of Chemo-Biosensing, Key Laboratory of Functional Molecular number, as well as fast ligand exchange rates.2
2,23
Cerium is
Solids, Ministry of Education, College of Chemistry and Materials Science, Anhui
a special among lanthanides because it can form a tetravalent
oxidation state under aqueous solutions. Ce(IV) ion has been
found to possess striking activity in promoting bis(4-
nitrophenyl) phosphate (BNPP) hydrolysis, which was
Normal University, Wuhu, 241000, P. R China. E-mail: dujinyn@mail.ahnu.edu.cn;
zhucq@mail.ahnu.edu.cn
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/d0ra07429b
1
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attributed to the ability of Ce(IV) to form covalent bonds with chemical reagents were used without any treatment. All solu-
phosphate substrates and promote the formation of penta- tions were prepared with ultrapure water (18.25 MU cm).
24,25
coordinate intermediates.
Many Ce(IV)-based mimetic
hydrolase models have been applied in the study of phosphate
ester hydrolysis. It was reported that using remarkable speci-
2
.2 Apparatus
The morphology of the as-prepared CeCDs was characterized by
transmission electron microscopy (TEM, JEOL 2010, Japan). X-
ray photoelectron spectroscopy (XPS) was employed to investi-
gate the content of elements and the conformation of the
CeCDs was determined by Thermo Scientic K-Alpha instru-
ment. Elemental analysis was carried out by energy dispersive X-
ray spectroscopy (EDS) conducted at the accelerating voltage of

city of Ce(IV)/EDTA complex, gap-site in substrate DNA can be
26
selectively hydrolyzed. Moreover, the dicerium complexes
were proved to be able to carry out the double strand hydrolysis
of DNA. Recently, Ceria (CeO
27
2
) nanocrystals were served as
mimic phosphatases for cleaving the phosphate ester bond in
28
para-nitrophenyl phosphate. However, owing to the poor
water-solubility and complicated synthesis-processing of these
complexes containing metallomicelles or macrocyclic ligand,
the application of these articial hydrolytic metalloenzymes
catalytic systems is largely restricted.
200 kV on a Philips S-4800 instrument. UV-vis spectra of the
samples were obtained on a Hitachi UV-2910 spectrophotom-
eter. The uorescence spectra were recorded with an Edinburgh
FS5 uorescence spectrophotometer. Fourier transform
infrared (FTIR) spectra were carried out on a PerkinElmer PE-
983 FTIR spectrophotometer. The degradation of chlorpyrifos
was employed by Gas Chromatography-Mass Spectrometer (GC-
MS Thermo Trace-1300 ISQ-Mass). Phosphate ion were quanti-
Fortunately, uorescent carbon dots (CDs) with the advan-
tages of optimal size, biocompatibility, and good photo- or
29
electro-catalytic efficiency, have been applied in many elds
including bioimaging and sensing, nanomedicine, optoelec-
30
tronic devices, ion detection and catalysis. More and more
carbon-based nanomaterials were demonstrated to hold an

ed using a ICS-1100 ion chromatography (Thermo, USA)
31
equipped with an AS11-HC column (250 mm ꢀ 4 mm) and
intrinsic enzyme-like activity. Hitherto, most of the catalytic
3
2,33
a conductivity detector (883 Basic IC plus 1). The mobile phase
reactions of nanozymes are based on peroxidase,
ꢁ
1
34,35
36,37
38,39
was 30 mM sodium hydroxide at a ow rate of 1.2 ml min and
the injection volume of 25 ml.
oxidase,
laccase,
and catalase,
etc. However, articial
hydrolases based on carbon nanomaterial are also highly
desirable.
Herein, based on high sensitivities of P–O bond cleavage 2.3 Preparation of CeCDs
toward lanthanide ions and uorescence properties of CDs, we
The CeCDs were synthesized by a hydrothermal method. Typi-
cally, 1.0000 g of EDTA$2H O and 2.3451 g of Ce(NO ) $6H O
2 3 3 2
have successfully synthesized uorescent Ce-doped CDs
CeCDs) by a one-step hydrothermal method using EDTA as
a carbon source and Ce(NO as a Ce dopant. We used hydro-
lysis of phosphate diester as a model reaction for the cleavage of
phosphorus bond of DNA and RNA. Here, BNPP was chosen as
a DNA model substrate, and phosphatase-like activity of the
obtained CeCDs was explored based on the inner lter effect
(
were dispersed in 30 ml ultrapurewater under magnetic stirring
to form a transparent solution. Aerward, the mixture solution
was transferred into a 50 ml Teon equipped stainless steel
3 3
)
40
ꢂ
autoclave and heated at 180 C for 6 h. Aer the resultant
solution was cooled to room temperature naturally, the trans-
parent yellow solution was ltered and as-collected supernatant
was further dialyzed in the dialysis bag (molecular weight: 1000
kDa) for 24 h to remove the excess reactants. The puried
(IFE), i.e., the maximum absorption range of BNPP hydrolysis
products (p-nitrophenol) partly overlaps with emission spec-
trum of the as-prepared CeCDs, resulting in an efficient uo-
rescence quenching of the CeCDs. The rate of BNPP hydrolysis
was calculated and the possible catalytic mechanism was
proposed. Our results suggested that the uorescence CeCDs
can promote and monitor the process of phosphate ester
ꢁ1
ꢂ
CeCDs (concentration: 15.8 mg ml ) were preserved at 4 C for
the following experiments.
2.4 Phosphatase activity of CeCDs
hydrolysis cleavage. Finally, the CeCDs was further applied to The disodium salt of BNPP was used as a DNA model substrate
degradation of organophosphorus pesticide chlorpyrifos. to investigate phosphodiesterase activity. Commonly, in the
Within 48 h, the degradation efficiency was estimated to be presence of phosphatase, BNPP was transformed into p-nitro-
74.50%.
phenol which induced the increase in the absorption of the
band at 400 nm. Here, the as-prepared CeCDs were deemed as
articial phosphatases to investigate the catalytic activity of
BNPP hydrolytic cleavage. In a typical experiment, 1.0 ml BNPP
solution (5 mM), 0.5 ml CeCDs and 1.0 ml Tris–HCl buffer were
2
. Experiments
2.1 Materials and reagents
Cerium(III) nitrate hexahydrate (Ce(NO
3
)
3
$6H
2
O, 99.99%, Ada- introduced into a colorimetric tube. The color of the solution
mas), ethylenediaminetetraacetic acid disodium salt dihydrate changed gradually from transparent to yellow as the reaction
EDTA$2H
O, 98%) was acquired from Guoyao Chemical proceeding. The process of BNPP hydrolysis was detected
(
2
Reagent Company (Shanghai, China), BNPP was prepared from spectrophotometrically by monitoring the time evolution of p-
the commercial product purchased from Sigma. Chlorpyrifosv nitrophenol (lmax ¼ 400 nm) through a wavelength scan from
ꢂ
(
>99%) was obtained from Aladdin. Tetracosane, acetone, ethyl 200 to 900 nm at 25 C, containing 100 equivalents of substrate
acetate and trichloromethane were obtained from Aladdin. All relative to the catalyst, until roughly 2% reaction conversion was
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reached. Comparative experiments in the absence of the CeCDs demonstrates the emission spectra of CeCDs excited by the light
were carried out under identical conditions.
of wavelength ranging from 320 to 400 nm. The as-prepared
CeCDs have the strongest emission at 426 nm when excited at
350 nm (lex). It could also be observed that the CeCDs exhibit
excitation-independent emission properties (the uorescent
emission spectra of Ce-free bare CDs is shown in Fig. S2† for
comparison), which could be attributed to the homogeneous
2.5 Degradation of pesticide
The degradation rate of pesticides catalyzed by CeCDs in
acetone was measured by GC-MS, using chlorpyrifos as an
external standard. Specic operations and implementation
processes are detailedly described in ESI.†
44,45
surface structure, and monodispersity of the CeCDs.
In
addition, the average uorescence quantum yield (QY) of the
CeCDs in aqueous solution at room temperature was measured
to be 27% using quinine sulfate as a reference (QY of quinine
sulfate is 54% at 350 nm excitation wavelength), which is
3. Results and discussion
3.1 Characterization of CeCDs
46
comparable with that in the previous reported CDs. However,
the QY of the Ce-free bare CDs was only 13%, lower than that of
the CeCDs. The results indicate that the introduction of metal
The CeCDs were synthesized according to the protocol
described in the experimental section. The structure and
morphology of the CeCDs were analyzed using TEM. Fig. 1A
clearly shows that the resulted CeCDs are well mono-dispersed.
The high resolution transmission electron microscopy
47
Ce dramatically improves uorescent performance of CDs.
The FTIR spectrum of the CeCDs is shown in Fig. 2A.
ꢁ
1
Remarkably, the peak emerges at 3440 cm , corresponding to
(HRTEM) image (inset in Fig. 1A) displays a lattice fringe
ꢁ
1
the stretching vibration of O–H. The peak at 1679 cm is
attributed to C]O stretching vibration, and the stretching
spacing of 0.31 nm, which is attributed to the (002) spaces of
41
graphitic carbon. The size distribution ranges from 1.9 nm to
.5 nm (Fig. 1B) with an average size of around 2.7 nm (100
ꢁ1
vibration bands of C–O at 929 and 728 cm indicate the pres-
3
ence of carboxylic acid and other oxygen-containing functional
nanoparticles were counted), which is larger than that of the Ce-
free bare CDs (an average diameter 2.3 nm, Fig. S1†).
ꢁ
1
groups. The absorbance at 1630 cm
is assigned to N–H
bending vibration, suggesting the existence of amino-
containing functional groups. These peaks suggest that the
CeCDs have logical hydrophilic properties as well as signicant
As shown in Fig. 1C, optical absorption in the UV region was
observed in the UV-vis absorption spectrum of CeCDs. The
CeCDs have two strong peaks at 250 and 280 nm, corresponding
48
stability and dispersibility in water.
42,43
to p–p* and n–p* transitions, respectively.
From the inset of
The elemental composition of the CeCDs was analyzed using
X-ray spectroscopy (EDS). EDS spectrum of the CeCDs presents
Fig. 1C, it was observed that the CeCDs were yellow liquid
illuminated with ambient light, and the CeCDs emitted bright
blue light under the UV light excitation (365 nm). Fig. 1D

ve elements of C, N, O, Ce and Na from 0 to 10 keV (Fig. S3†), in
Fig. 1 (A) TEM and HRTEM (inset) images. (B) Size distribution of the as-prepared CeCDs. (C) UV-vis absorption spectra of the CeCDs solution.
Inset: (a) Photograph of CeCDs illuminated with ambient light, (b) photograph of CeCDs under UV light (365 nm). (D) Fluorescence emission
spectra of the CeCDs dispersed in water (ex: 320–400 nm, em: 350–650 nm).
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Fig. 2 (A) FTIR spectrum, (B) XPS entirety and high-resolution survey of (C) C 1s, (D) N 1s, (E) O 1s and (F) Ce 3d of the CeCDs.
ꢂ
good agreement with those of the precursors. Moreover, the XPS and no catalyst aer incubation for 3 h at 25 C. No obvious
spectrum analysis show that C1s, N1s, O1s and Ce3d peaks locate absorption at 400 nm was observed when Ce-free bare CDs were
at 284.4, 407.4, 532.1 and 890.0 eV (Fig. 2B), respectively. Since served as the catalyst or no catalyst was presented for the
there are no other carbon sources, carbon elements in the hydrolytic cleavage of BNPP. At variance, in the presence of
CeCDs must be from the EDTA, while nitrogen and cerium are CeCDs, remarkable absorption peak at 400 nm was caught
49
from the introduction of Ce(NO ) . The high resolution XPS along with the appearance of light yellow color (c of inset in
3
3
spectrum of C1s exhibits C]C/C–C, C–O/C–N/C]N, C]O main Fig. 3). These results clearly demonstrate that the CeCDs have
peaks at the corresponding binding energies of 284.4, 285.6 and an obvious phosphatase-like activity.
5
0
2
88.8 eV (Fig. 2C). In the high-resolution region of N1s as
shown in the Fig. 2D, there are three peaks residing at 399.5, cerium source in combination with EDTA, temperature and
00.0 and 407.4 eV, which are attributed to the C–N–C, (C3)–N, time of the reaction, can affect phosphatase-like activity of the
It was found that several factors, including concentration of
4
45
N–H groups, respectively. The O1s spectrum can be resolved as CeCDs. To optimize the synthesis conditions of the CeCDs, rst,
follows: C]O is at 533.1 eV, C–OH/C–O–C is at 532.1 eV the ratio of EDTA to cerium nitrate was controlled from 0.15 to
Fig. 2E). The Ce 3d spectrum consists of four characteristic 0.75, maintaining other parameters such as reaction time (4 h)
peaks (Fig. 2F). The peaks at 903.9 and 899.6 eV are ascribed to and temperature (200 C) as constants. Aer purication, the
the binding energy of Ce 3d3/2 orbital, while the peaks at 885.6 CeCDs with different ratio of EDTA to cerium nitrate were added
and 881.1 eV correspond to the binding energy of Ce 3d5/2 to 1 ml BNPP (5 mM) for incubation 2 h at 25 C. The absor-
51
(
ꢂ
ꢂ
orbital. Besides, the peak positions of the Ce 3d orbitals and the bance increase value of the p-nitrophenol band at 400 nm was
binding energy difference (DE) between these two components
were approximate, indicative of an identical 3+ valence state of
52,53
Ce.
In addition, the FTIR and XPS show that there are rich
oxygen-containing groups and amino groups on the surface of
54–56
the CeCDs, indicative of a good hydrophilicity.
tates catalytic hydrolysis of BNPP.
This facili-
3.2 Optimization of synthesis and hydrolysis conditions for
phosphatase-like activity of CeCDs
The phosphatase-like activity of CeCDs was investigated by
employing the substrate BNPP in aqueous solution. Hydrolysis
product of BNPP, p-nitrophenol, exhibits distinct optical
absorption properties (lmax ¼ 400 nm), making it possible to
track the reaction progress by using absorbance spectroscopic
means. Fig. 3 shows the UV-vis spectra through a wavelength
scan from 300 to 600 nm collected in the process of hydrolytic
Fig. 3 The UV-vis absorption spectra for BNPP, BNPP + Ce-free bare
CDs and BNPP + CeCDs solution after incubation for 3 h at 25 C.
Inset: The photograph of the corresponding system under ambient
ꢂ
cleavage of BNPP in the presence of CeCDs, Ce-free bare-CDs conditions: (a) BNPP, (b) BNPP + Ce-free bare CDs, (c) BNPP + CeCDs.
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selected as a standard for evaluating phosphatase-like perfor- 0.9978), where A
mance, which is also a criterion for evaluating optimization of 2 days at 25 C, A is the absorbance of 400 nm at time t.
N
is the absorbance at 400 nm aer incubation
ꢂ
57,58
The
t
the following other synthesis conditions. As is observed in release of the rst p-nitrophenol molecule from BNPP is the
Fig. S4A,† at the optimum ratio of 0.5 between EDTA and cerium rate-determining step in the BNPP catalytic hydrolysis. There-
nitrate, the CeCDs show the highest catalytic properties. Then, fore, apparent rate constant, kobs, is almost equal to the rst
18
the effect of reaction temperature on the catalytic properties of order rate constant, kcat
.
For comparison, the same concen-
as-synthesized CeCDs was also investigated. The reaction tration of Ce-free bare CDs was added to BNPP substrate, then
ꢂ
temperature increases in a step of 20 from 140 to 220 C with the absorption spectra were measured at 20 min intervals for

xing the ratio of EDTA to cerium nitrate of 0.5. The results 2 h. From the Fig. S6,† there was no obvious change in the
ꢂ
indicate that the CeCDs synthesized at 200 C exhibit a higher absorption spectrum. The results on the one hand indicate that,
phosphatase-like activity (Fig. S4B†). Similarly, the duration of Ce-free bare CDs have no signicant catalytic activity for the
the reaction was also adjusted to estimate the optimum reaction hydrolysis of BNPP, and on the other hand, the element Ce is
time. As illustrated in Fig. S4C,† the CeCDs synthesized with 6 h essential and plays a vital role in catalyzing the hydrolysis of
reaction display the highest catalytic activity, implying that 6 h BNPP.
of reaction time is the optimum experimental condition.
In order to eliminate the possibility of photocatalysis, the
Therefore, under the optimal conditions (i.e., the ratio of EDTA absorbance spectra of BNPP upon the addition of CeCDs under
ꢂ
to cerium nitrate: 0.5, reaction temperature: 200 C, reaction dark conditions were presented in Fig S7.† It can be observed
time: 6 h), a facile carbonization and efficient Ce-doping occurs that there is a signicant increase in absorption at 400 nm
to generate the CeCDs with appreciable phosphatase-like within 120 min. From the plot of log[A /(A ꢁ A )] values versus
N
N
t
2
activity.
time t (Fig. S7B,† R ¼ 0.9950), the pseudo-rst-order-rate
Then, the pH of BNPP hydrolysis was optimized in the range constant of BNPP hydrolysis was calculated to be kcat ¼ 2.6 ꢀ
ꢁ3
ꢁ1
ꢁ5
of 5–10 in Tris–HCl (0.05 M) buffer solution. As shown in 10 min (error ¼ 5.5 ꢀ 10 ), which is basically equal to that
Fig. S5,† the catalytic cleavage activity of CeCDs for BNPP of under sunlight. These results indicate that photocatalysis is
hydrolysis shows the best performance in pH 8.2–8.7. There- not responsible for the hydrolysis of BNPP, thus the CeCDs
fore, in the subsequent experiments, pH 8.5 was used as the indeed serves as a mimic enzyme in the hydrolysis reaction.
optimal condition for BNPP hydrolysis.
Furthermore, potassium persulfate was added to the catalytic
system as an electron scavenger, and the absorption spectra
were recorded (Fig. S8A†). It can be observed that the absorption
at 400 nm increases gradually, and the calculated pseudo-rst-
3.3 Catalytic kinetics and indication process for BNPP
hydrolysis
ꢁ3
ꢁ1
order-rate constant of BNPP hydrolysis, kcat ¼ 2.5 ꢀ 10 min
The efficiency of CeCDs for hydrolytic cleavage of BNPP in
aqueous solution was determined spectrophotometrically by
monitoring the time evolution of p-nitrophenol (labs ¼ 400 nm)
ꢁ4
(
error ¼ 3.02 ꢀ 10 ), is consistent with the result without
adding potassium persulfate (Fig. S8B†), indicating that there
was no electron transfer in the catalytic process.
ꢂ
through a wavelength scan from 300 to 600 nm at 25 C, until
To further understand the acceleration behavior of CeCDs on
the hydrolysis of BNPP, the uorescence emission spectra of the
CeCDs were determined in the presence of different concen-
tration BNPP. As shown in Fig. S9,† the uorescence intensities
of the CeCDs decrease with the increase in BNPP concentration.
This observation indicates a strong binding between the CeCDs
and BNPP molecules, which leads to the photoelectron transfer
roughly 2% reaction conversion was reached. The absorbance
spectra of BNPP upon the addition of CeCDs were shown in
Fig. 4A, where the spectra were recorded at 10 min intervals for
2
h. From the Fig. 4A, the absorption at 400 nm increases
gradually with the appearance of yellow solution. The pseudo-
rst-order-rate constants of BNPP hydrolysis, kcat ¼ 1.84 ꢀ

ꢁ3
ꢁ1
ꢁ5
10
min (error ¼ 3.89 ꢀ 10 ), were obtained directly from
59
from BNPP to the CeCDs. Because oxygen atom of the BNPP
2
the plot of log[A
N
/(A
N
ꢁ A )] values versus time t (Fig. 4B, R ¼
t
Fig. 4 (A) Time-dependent UV-vis spectral changes in BNPP solution catalyzed by CeCDs. The spectra were recorded at intervals of 10 min. The
inset is the color change of photographs of BNPP solution in the presence of CeCDs in natural light. (B) Plots of log[A
the catalytic hydrolytic cleavage of BNPP at room temperature.
N
/(A
N
ꢁ A
t
)] versus time for
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molecule has a strong affinity to metallic ions, the phosphoryl above step, rapidly releases phosphoric acid and another p-
oxygen of BNPP molecule may bond with the metal ion (Ce(III)) nitrophenol and simultaneously regenerates the active species
of CeCDs when the BNPP was added into the CeCDs solution, of step 1 (the product of phosphate ion has been caught by ion
which is proved by the obvious change of uorescence intensity chromatography, see Fig. S10†). Ultimately, when another BNPP
of the CeCDs in Fig. S9.† This situation could promote the molecule is associated with the regenerated metal hydroxide,
formation of the reaction intermediate with BNPP and the the catalytic process begins to recycle. Our proposed mecha-
metal-bound hydroxide activation, which can facilitate an nism is similar to that of BNPP hydrolysis catalyzed by lantha-
61
intramolecular nucleophilic reaction. As is known, the reaction nide or transition metalloenzymes.
rate of intramolecular reactions can be considerably enhanced The phosphatase catalytic behavior of CeCDs was further
60
compared with that of intermolecular reactions. Thus, investigated according to the steady-state kinetic analysis using
because of the synergic effects of Ce(III) ion and metal hydroxide BNPP as the substrate. The kinetic data were obtained by
activation, the P–O bond of BNPP molecule can be cleaved more varying substrate concentration while keeping the other
62,63
favorably.
substrate concentration constant.
It can be observed in
According to the above analysis, the possible reaction Fig. S11A† that the hydrolytic reaction of phosphate ester
mechanism for the catalytic hydrolysis of BNPP was proposed as catalyzed by the CeCDs was heavily dependent on the concen-
shown in Scheme 1. In the step 1, due to a good hydrophilicity, tration of substrate which followed a typical Michaelis–Menten
64
the metal Ce on the surface of CeCDs in aqueous solution are behavior towards BNPP. Hence, a series of catalytic reaction
favorably bonded with water molecules. Under alkaline condi- parameters could be calculated according to the Line weaver–
tions, the deprotonation of the metal-bound water forms Burk double reciprocal plot (Fig. S11B†) based on the equation:
a nucleophilic metal hydroxide. In the step 2, the intermediate 1/v ¼ (K /V ) ꢀ (1/[S]) + 1/vmax, where v is the initial reaction
m
max
m
(R) is formed by coordination of the oxygen atom of BNPP with velocity, K is the Michaelis–Mentenconstant, vmax is the
the aqua-hydroxyl active species, which is benecial to the maximum reaction velocity, and [S] is the concentration of
transformation of an intermolecular reaction into an intra- substrates. In Table S1,† we compare our kinetic parameters
molecular one. In the step 3, intramolecular metal hydroxides result to those reported in the literatures on the other catalysts.
m
attack the positive-charged P atoms of the BNPP molecule, The apparent K value of CeCDs with BNPP as the substrate
promoting the release of p-nitrophenol and production of (0.866 mM) is lower than that of metal complexes for mimetic
phosphate monoester with rst-order-rate constant (the rate- phosphate hydrolases, suggesting that the CeCDs is an excellent
65,66
determining step). And in the step 4, p-nitrophenyl phosphate mimetic hydrolase with a higher affinity.
(NPP), which is one of the products of BNPP hydrolysis in the
Simultaneously, we measured the uorescence behavior of
CeCDs upon the addition of BNPP until roughly 2% reaction
conversion was reached, where the spectra were recorded for 2
hours at 10 min intervals. As shown in Fig. 5A, the uorescence
of CeCDs was gradually quenched with the appearance of dark
blue color, corresponding to the increase of absorption peak at
400 nm in UV-vis absorption spectra (Fig. 4A). This scenario
could provide a basis for quantitative monitoring of the catalytic
hydrolytic cleavage of BNPP. In order to comprehend the uo-
rescence quenching mechanism of CeCDs, the emission spectra
of CeCDs were found to overlap partially with the absorption
spectra of p-nitrophenol (Fig. S12†). Note that p-nitrophenol can
shield the emission light of the CeCDs, therefore, the uores-
cence quenching mechanism may be regarded as uorescence
67,68
resonance energy transfer (FRET) or IFE.
However, the fact
that there is no change in the uorescence lifetime (Fig. S13†) of
CeCDs before and aer adding BNPP indicates that no FRET
process occurs between CeCDs and p-nitrophenol. These results
suggest that the highly efficient IFE is the dominant reason for
the uorescence quenching of CeCDs in the catalytic system.
Moreover, the rate constants of the catalytic hydrolytic
cleavage of BNPP was calculated from the uorescence spectra
6
9
of the CeCDs mimetic hydrolase. The plot of log[F /(F ꢁ F )]
0
0
t
versus time t was shown in Fig. 5B. Here, F is the initial uo-
0
rescence intensity of the CeCDs at maximum emission, and F
t
represents the uorescence intensity of the CeCDs at maximum
emission at time t. The rate constant (kcat) of BNPP hydrolysis
calculated from the linear relationship of log[F
0
/(F
0
ꢁ F
t
)] versus
Scheme
1 Schematic illustration of catalytic hydrolysis cleavage
ꢁ3
ꢁ1
mechanism of BNPP catalyzed by the fabricated CeCDs.
time t was 1.33 ꢀ 10
min , which is approximately
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Fig. 5 (A) Time-dependent fluorescence emission spectral changes of the CeCDs in BNPP solution. The spectra were recorded at intervals of
0 min, excitation wavelength ¼ 350 nm. The inset is the fluorescence color photographs of CeCDs solution in the presence of BNPP under
a 365 nm UV lamp. (B) Plots of log[F /(F )] versus time for the catalytic hydrolytic cleavage of BNPP at room temperature.
ꢁ F
1
0
0
t
Fig. 6 (A) Time-dependent GC-MS chromatogram of chlorpyrifos samples upon addition of the CeCDs. Retention time: 19.91 min. (B) Plots of ln
1/(1 ꢁ degradation efficiency)] versus time for the catalytic degradation of chlorpyrifos at room temperature.
[
consistent with the values calculated from relative intensity of CDs was also investigated. As shown in Fig. S14,† the change in
absorbance. All of the experimental results suggest that the peak area was negligible, indicating that the Ce-free bare CDs
catalytic hydrolysis process of BNPP can be monitored by the have no obvious catalytic performance for the chlorpyrifos

uorescence behavior of mimic enzyme itself. Therefore, it is degradation. These results again verify that the element of Ce is
undoubtedly convenient for a class of reaction without specic indispensable for cleavage of the P–O bond.
spectrophotometric characteristics of the substrates.
4. Conclusion
3.4 Pesticide chlorpyrifos degradation
To validate the availability of the established catalytic systems, In summary, we have prepared water-soluble, uorescent
the CeCDs were further applied for the degradation of pesticide CeCDs by a facile one-step hydrothermal method using EDTA
chlorpyrifos. The degradation rate of chlorpyrifos was analyzed as a carbon source and Ce(NO ) as a Ce dopant. Using BNPP
3 3
by GC–MS using chlorpyrifos as the external standard. As shown as a DNA model substrate, phosphatase-like activity of the as-
in Fig. 6A, the peak area values are along with a signicant obtained CeCDs were systematically investigated. On the one
decrease within 48 h. The natural logarithm of the concentra- hand, a highly catalytic performance toward BNPP hydrolysis
tion of chlorpyrifos vs. time was plotted (Fig. 6B). The rst order was found due to the synergistic effect of Ce(III) ion and metal
ꢁ2
2
rate constant was calculated to be 3.1 ꢀ 10 h (R ¼ 0.996), hydroxide activation; on the other hand, IFE leads to uo-
indicative of an excellent degradability of CeCDs for chlorpyr- rescence change of the CeCDs mimetic hydrolase in the
70
ifos. The degradation rate of chlorpyrifos by CeCDs within process of BNPP hydrolysis. Moreover, the values of the rate
8 h was 74.50% (Table S2†). Herein, the degradation constants calculated from the absorbance change of p-nitro-
percentage of chlorpyrifos was obtained according to the phenol are primarily consistent with those from the uores-
4
71
following formula: Percentage of chlorpyrifos degradation ¼ cence intensity change of CeCDs mimetic hydrolase. These
(
peak area of initial concentration chlorpyrifos ꢁ peak area of unique properties would provide an ideal platform for in situ
chlorpyrifos)/peak area of initial concentration chlorpyrifos. As monitoring the catalytic process of hydrolysis of phosphate
a comparison, degrading chlorpyrifos catalyzed by Ce-free bare diester.
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2
0 P. Gomeztagle and A. K. Yatsimirsky, Inorg. Chem., 2001, 40,
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Conflicts of interest
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There are no conicts to declare.
3
2
2 H.-l. Shang, L. Yu, S. Li and J.-q. Xie, J. Dispersion Sci.
Technol., 2014, 35, 411–417.
Acknowledgements
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