off-On fluorescent sensing of organophosphate pesticides using a carbon dot-Au(III) complex

Article abstract of DOI:10.1039/c7ra13404e

Herein, we report novel off-on fluorescent sensing of organophosphate pesticides using a carbon dot (CD)-Au(iii) complex/acetylcholinesterase (AChE) system. The above sensor utilizes the quenching of CD fluorescence by Au(iii) and its subsequent recovery by thiocholine, which is generated by AChE-catalyzed hydrolysis of acetylthiocholine (ATCh) and effectively scavenges Au(iii). In the presence of organophosphates, the catalytic activity of AChE is inhibited, allowing these species to be quantified based on the decreased recovery of CD fluorescence intensity. The developed sensor was used to analyze a real pesticide-spiked sample (4.48 μM), achieving a recovery of 99.85% and exhibiting a linear response range of 0.45-44.80 μM.

Full text of DOI:10.1039/c7ra13404e

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“Off–On” fluorescent sensing of organophosphate
pesticides using a carbon dot–Au(III) complex†
Cite this: RSC Adv., 2018, 8, 11551
ab
ab
Minmin Li,ab Jia Lu,ab Bei Fan,
Yan He,ab Yatao Huangab
*
Miao Wang,
abc
*
and Fengzhong Wang
Herein, we report novel “off–on” fluorescent sensing of organophosphate pesticides using a carbon dot
(CD)–Au(III) complex/acetylcholinesterase (AChE) system. The above sensor utilizes the quenching of CD
fluorescence by Au(III) and its subsequent recovery by thiocholine, which is generated by AChE-catalyzed
hydrolysis of acetylthiocholine (ATCh) and effectively scavenges Au(III). In the presence of
organophosphates, the catalytic activity of AChE is inhibited, allowing these species to be quantified
based on the decreased recovery of CD fluorescence intensity. The developed sensor was used to
analyze a real pesticide-spiked sample (4.48 mM), achieving a recovery of 99.85% and exhibiting a linear
response range of 0.45–44.80 mM.
Received 18th December 2017
Accepted 19th March 2018
DOI: 10.1039/c7ra13404e
rsc.li/rsc-advances
an effective Au(III) scavenger) generated by AChE-catalyzed
hydrolysis of acetylthiocholine (ATCh) that is structurally
1 Introduction
similar to ACh. In the presence of organophosphates, the
catalytic activity of AChE is inhibited, allowing these species to
be quantied based on the decreased recovery of CD uores-
cence intensity.
Organophosphate pesticides nd extensive applications world-
wide, exhibiting acute toxicity upon ingestion and thus posing
a serious risk to human health.1,2 The above pesticides mainly
target acetylcholinesterase (AChE),3 inhibiting its catalytic
activity and thus hindering the hydrolysis of acetylcholine (ACh,
a neurotransmitter), with the accumulation of excess ACh
leading to lethal overexcitation.4,5 However, the above activity
inhibition can be successfully utilized in organophosphate
pesticide sensing.
Carbon dots (CDs) have recently found numerous applica-
tions due to exhibiting excellent uorescence, good biocom-
patibility, and low toxicity and cost.6,7 Recently, much effort has
been directed at utilizing CD uorescence or electro-
chemiluminescence to fabricate “off–on” or “on–off”
sensors,8–10 which exhibit the advantages of fast response, easy
operation, and clear signal recognition.11 Notably, metal ions
such as Cu2+, Fe3+, Hg2+, K+, and Ag+ (ref. 11–15) are able to
quench the uorescence of CDs,16 allowing the sensing of heavy
metals, organic pollutants in water, sugar in blood, and
biomarkers in living cells.17–20
2 Materials and methods
2.1 Materials and reagents
Eggs were purchased from the Chaoshifa supermarket. AChE
and ACh were purchased from Sigma-Aldrich (USA). H2SO4,
NaOH, and HAuCl4 were sourced from Sinopharm Co. (China),
and all other chemicals were obtained from the Beijing Chem-
ical Plant. Deionized (DI) water was produced using the Milli-Q
system (USA). Incubators and magnetic stirrer plates were
purchased from IKA Instrument Co. (Germany). Fluorescence
spectra were recorded on a HITACHI 2000 spectrometer (Hita-
chi Co., Japan), and Fluorescence Spectrometer FS5 from
Edinburgh Instruments (U.K.). Transmission electron micros-
copy (TEM) imaging was performed using a JEM-2100 instru-
ment (JEOL Co., Japan). The Fourier transform infrared
spectrometer was Tensor 27 from Buruker. Co. etc.(Germany). X-
ray Photoelectron Spectroscopy (XPS) was tested by using PHI
Quantera SXM, from ULVAC-PHI (Japan).
Herein, we report a novel “off–on” uorescent biosensor for
organophosphate pesticides, in which the CD uorescence
initially quenched by Au(III)20 is recovered by thiocholine (TCh,
2.2 CD synthesis
aKey Laboratory of Quality & Safety Risk Assessment on Agro-Products Processing
(Beijing), Ministry of Agriculture, Beijing 100193, China. E-mail: fanbei517@163.
com; wangfengzhong@sina.com
A procedure previously reported elsewhere23 was optimized and
used to prepare CDs.
bInstitute of Food Science and Technology, Chinese Academy of Agricultural Sciences,
Fresh egg white (4 mL) was dissolved in DI water (40 mL)
upon 3 min stirring, and a 10 mL aliquot of the obtained
solution was transferred into a 50 mL ask, treated with 98 wt%
H2SO4 (4 mL, slow addition!), agitated, and placed in a water
Beijing 100193, China
cTibet Academy of Agricultural and Animal Husbandry Sciences, Lhasa 850000, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra13404e
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bath (50 ꢀC) for 2 h. Subsequently, the mixture was neutralized
with 5 M aqueous NaOH and subjected to 5 min centrifugation
at 5000 rpm. The supernatant was transferred into an Eppen-
dorf tube and treated with dichloromethane (3 mL), with
another 5 min centrifugation at 5000 rpm resulting in the
extraction of the synthesized uorescent carbon dots into the
organic phase. The dichloromethane phase was separated and
blown dry with nitrogen, and the thus obtained CDs were
dispersed in DI water for utilization in the next step.
2.3 Characterization of CDs and CD/Au(III) systems
The emission spectrum of carbon dots was scanned with
different excitation wavelength (slit width ¼ 10 nm). And the
morphology of carbon dots was characterized by transmission
electron microscope (TEM). The surface groups and elemental
speciations were tested by FTIR and XPS respectively.
A series of Eppendorf tubes was charged with the above-
mentioned aqueous CD dispersion (20 mL), which was followed
by the addition of 0.2 M phosphate-buffered saline (PBS, pH 7.0,
100 mL) and solutions containing different concentrations of
Au(III) ions. The resulting mixtures were diluted with DI water to
2 mL, incubated at room temperature for 8 min, and probed by
uorescence spectroscopy.
Scheme 1 Mechanism of organophosphate pesticide sensing by the
developed CD/Au(III) sensor.
(S.2†) showed that the C, O, N and S were at present of 62.84,
12.53, 22.56 and 2.07%, respectively. The uorescence quantum
yield (QY) of carbon dot was 7.06%, tested by Fluorescence
Spectrometer FS5 (Edinburgh Instruments, U.K.). However, with
excitation light with different wavelength, the emission light
wavelength also varies. Regarding the steady of testing and the
harm of UV light to AChE, we choose 470 nm as the excitation
wavelength in following experiments.
2.4 Organophosphate pesticide sensing
AChE-catalyzed hydrolysis of ATCh afforded TCh that could
bind Au(III) ions and thus recover CD uorescence. Since
organophosphate pesticides inhibit AChE, their presence
resulted in slower TCh generation and thus decreased CD
uorescence recovery. A series of organophosphate pesticide
solutions of different concentrations was added to the CD/
Au(III)/(ATCh + AChE) system to construct a calibration curve.
3.3 Fluorescence quenching by different metal ions
3 Results and discussion
3.1 Principle of “off–on” uorescent sensing
Metal ions exhibit a well-documented ability to quench the
uorescence of CDs. The results obtained for selected ions
(Fig. 2) revealed that the most signicant quenching was
observed for Au(III), with increasing Au(III) concentration thus
causing a decrease of CD uorescence.
When excitation was performed at 365 nm, the uorescence
changes could be observed by the naked eye, as shown in Fig. 3a.
Fig. 3b shows that Au(III) could effectively quench CD uorescence,
with the uorescence intensity at the maximum emission wave-
length linearly decreasing with increasing Au(III) concentration.
AChE catalyzes the hydrolysis of S-acetylthiocholine iodide to
TCh,22 which can effectively capture Au(III) and thus recover the
CD uorescence quenched by the same.20
As mentioned above, organophosphate pesticides inhibit
AChE, thus decreasing the rate of TCh generation and
hindering uorescence recovery, which was used for their
detection (Scheme 1).
3.2 CD synthesis and properties
3.4 Fluorescence recovery in the presence of ATCh and
selected anions
The synthesized CDs exhibited nearly spherical shapes (diameter
z 20–40 nm) and were well dispersible in DI water (Fig. 1a). With
the different excitation wavelength, the emission wavelengths of As mentioned above, AChE can catalyze the hydrolysis of S-
bare CDs vary from 480 to 530 nm, respectively (Fig. 1b). acetylthiocholine iodide into TCh23 that can effectively capture
According to the FITR spectrum (S.1†), the broad absorption Au(III) and thus recover CD uorescence.21
bands near 3400 cmꢁ1 was the character peak of n(O–H) and n(N–
The amount of produced TCh, and thus, the extent of CD
H). The peak at 1720 cmꢁ1 should be n(C]O). And the peak at uorescence recovery, was expected to be proportional to the
1600 cmꢁ1 should be scissor bending vibration of H2O. The quantity of added ATCh. A preliminary experiment was carried
peaks of 1395 cmꢁ1 and 1150 cmꢁ1 refer to the bending vibration out to prove feasibility of the mechanism. The naked CDs, CDs
of d(CH2) and n(C–O–H). Composition of carbon dots synthesized quenched by Au(III), CDs with Au(III) and ATCh, CDs with Au(III)
was analyses by using XPS. While insensitive to H, the result and ATCh hydrolyzed by AChE were compared by using
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Fig. 1 (a) TEM image of as-synthesized CDs dispersed in DI water (b) emission spectra of CDs varies with different excitation wavelength.
uorescent spectrum scanning. As Fig. 4 shown, the uores- that most inorganic ions and amino acids exhibited no
cence of CDs can be signicantly quenched by adding 50 mM apparent effects (Fig. 5). However, the addition of cysteine
Au(III). However, the CDs Au(III) ATCh hydrolyzed by AChE got signicantly increased the uorescence of the above system due
stronger uorescence as the TCh caught most of the Au(III). And to this compound possessing an SH group capable of scav-
it makes the uorescence of system just a little bit lower than enging Au(III), as outlined in Scheme 1.
naked CDs. Therefore, this experiment proved the assembled
scheme was correct.
3.5 Optimization of ATCh concentration
Next, we investigated the inuence of potential interferants
With the increasing of TCh coming from hydrolyzed ATCh,
more and more Au(III) were captured. However, this linear
on uorescence quenching in the CD/Au(III) system, showing
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Fig. 2 Fluorescence quenching effect of different ions.
Fig. 4 Fluorescence changes of CD-containing systems observed
upon the addition of Au(III).
Fig. 5 Influence of amino acids and common anions on fluorescence
quenching in the CD/Au(III) system.
mM) hydrolysis performed at 37 ꢀC for 8 min were added to the
CD/Au(III) system, and uorescence spectra were recorded aer
8 min (Fig. 6b). As a result, optimum performance was observed
at pH 6.98, which is similar to the pH of maximal AChE
activity.24
3.7 Effects of hydrolysis time
Fig. 3 (a) Visually observed changes of CD fluorescence (excitation at
365 nm) upon the addition of Au(III). (b) Dependence of fluorescence
intensity on Au(III) concentration.
The effect of hydrolysis time was investigated at pH 6.98. As
described above, the products of AChE-catalyzed ATCh (1 mL,
50 mM) hydrolysis performed at 37 ꢀC for 1, 3, 5, 8, 10, and
15 min were added to the CD/Au(III) system, and uorescence
spectra were recorded aer 8 min (Fig. 6c). The optimal
hydrolysis time was determined as 8 min and was used in
subsequent experiments.
dependence was only observed in a certain range. It can be
observed in Fig. 6a that the certain range was 0–25 mM. Thus,
the optimal ATCh concentration in the reaction system was
determined as 25 mM.
3.6 Effects of pH
3.8 Determination of organophosphate pesticides
Optimal concentrations of Au(III) and CDs were selected to The relationship between uorescence recovery and organo-
investigate the effect of pH, which was adjusted by PBS. phosphate pesticide concentration (Fig. 6d) was used to
Subsequently, the products of AChE-catalyzed ATCh (1 mL, 50 construct a calibration curve (Fig. 7).
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Fig. 6 Optimization of enzymatic hydrolysis parameters for better sensing. Effects of (a) ATCh concentration, (b) pH, and (c) hydrolysis time on
sensor fluorescence recovery. (d) Inhibition of sensor fluorescence recovery by different concentrations of organophosphate pesticides.
complex solution was added into tubes as redissolution liquid.
Subsequently, AChE and ATCh were added, and the mixture was
incubated for 8 min and treated with CD/Au(III), followed by
uorescence recovery measurements.
Then according to the correction curve established in 3.8, the
concentration of organophosphate can be obtained by calculation.
The obtained data were used to determine the pesticide
concentration and recovery as 4.47 mM and 99.85%, respec-
tively, using the abovementioned calibration curve.
The obtained recovery met the requirements of organophos-
phate pesticide determination, lying in the stipulated range of 80–
120%.
Fig.
7
Calibration
curve
for
organophosphate
pesticide
determination.
4 Conclusions
3.9 Organophosphate pesticide sensing in real samples
Herein, CDs prepared from egg white by a hydrothermal
method were used to fabricate a biosensor for detecting
organophosphate pesticides. Initially, the uorescence of CDs
was quenched by a certain amount of Au(III) ions, which were
subsequently scavenged by TCh generated by AChE-catalyzed
hydrolysis of ATCh, which resulted in CD uorescence
recovery. The activity of AChE was reduced in the presence of
organophosphates, which decreased uorescence recovery and
allowed the pesticide concentration to be determined using
a pre-established calibration curve. Tests performed for a real
An apple was homogenized and spiked with an organophos-
phate pesticide (4.48 mM) to afford a positive sample. The equal
volume of methanol was added into the spiked sample and
stirred violently for sufficiently extraction. Then the samples
with extraction solution were centrifuged for 5 min, and the
precipitation was discarded. Aer nitrogen blowing with Ter-
movap Sample Concentrator about 10 min, the methanol was
nearly removed. And equal volume of 10% ethanol–PBS
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pesticide-spiked sample (4.48 mM) indicated a recovery of
99.85%, revealing that the developed method is well suited for
practical applications, with its linear response range corre-
sponding to 0.45–44.80 mM.
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Abbreviation
AChE
ATCh
CD
Acetylcholinesterase
Acetylthiocholine
Carbon dot
ACh
TCh
DI
Acetylcholine
Thiocholine
Deionized
TEM
PBS
Transmission electron microscopy
Phosphate-buffered saline
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