A label-free silicon quantum dots-based photoluminescence sensor for ultrasensitive detection of pesticides

Article abstract of DOI:10.1021/ac403257p

Sensitive, rapid, and simple detection methods for the screening of extensively used organophosphorus pesticides and highly toxic nerve agents are in urgent demand. A novel label-free silicon quantum dots (SiQDs)-based sensor was designed for ultrasensitive detection of pesticides. This sensing strategy involves the reaction of acetylcholine chloride (ACh) with acetylcholinesterase (AChE) to form choline that is in turn catalytically oxidized by choline oxidase (ChOx) to produce betaine and H₂O₂ which can quench the photoluminescence (PL) of SiQDs. Upon the addition of pesticides, the activity of AChE is inhibited, leading to the decrease of the generated H ₂O₂, and hence the PL of SiQDs increases. By measuring the increase in SiQDs PL, the inhibition efficiency of pesticide to AChE activity was evaluated. It was found that the inhibition efficiency was linearly dependent on the logarithm of the pesticides concentration. Consequently, pesticides, such as carbaryl, parathion, diazinon, and phorate, were determined with the SiQDs PL sensing method. The lowest detectable concentrations for carbaryl, parathion, diazinon, and phorate reached 7.25 × 10^-9, 3.25 × 10^-8, 6.76 × 10^-8, and 1.9 × 10^-7 g/L, respectively, which were much lower than those previously reported. The detecting results of pesticide residues in food samples via this method agree well with those from high-performance liquid chromatography. The simple strategy reported here should be suitable for on-site pesticides detection, especially in combination with other portable platforms.

Full text of DOI:10.1021/ac403257p

Article
pubs.acs.org/ac
A Label-Free Silicon Quantum Dots-Based Photoluminescence
Sensor for Ultrasensitive Detection of Pesticides
Yinhui Yi,† Gangbing Zhu,‡ Chang Liu,† Yan Huang,‡ Youyu Zhang,*,† Haitao Li,† Jiangna Zhao,†
and Shouzhuo Yao†,‡
†Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and
Chemical Engineering, Hunan Normal University, Changsha 410081, P. R. China
‡State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University,
Changsha 410082, P. R. China
S
* Supporting Information
ABSTRACT: Sensitive, rapid, and simple detection methods for the screening of
extensively used organophosphorus pesticides and highly toxic nerve agents are in
urgent demand. A novel label-free silicon quantum dots (SiQDs)-based sensor was
designed for ultrasensitive detection of pesticides. This sensing strategy involves the
reaction of acetylcholine chloride (ACh) with acetylcholinesterase (AChE) to form
choline that is in turn catalytically oxidized by choline oxidase (ChOx) to produce
betaine and H2O2 which can quench the photoluminescence (PL) of SiQDs. Upon
the addition of pesticides, the activity of AChE is inhibited, leading to the decrease of
the generated H2O2, and hence the PL of SiQDs increases. By measuring the increase
in SiQDs PL, the inhibition efficiency of pesticide to AChE activity was evaluated. It
was found that the inhibition efficiency was linearly dependent on the logarithm of
the pesticides concentration. Consequently, pesticides, such as carbaryl, parathion,
diazinon, and phorate, were determined with the SiQDs PL sensing method. The
lowest detectable concentrations for carbaryl, parathion, diazinon, and phorate reached 7.25 × 10−9, 3.25 × 10−8, 6.76 × 10−8,
and 1.9 × 10−7 g/L, respectively, which were much lower than those previously reported. The detecting results of pesticide
residues in food samples via this method agree well with those from high-performance liquid chromatography. The simple
strategy reported here should be suitable for on-site pesticides detection, especially in combination with other portable platforms.
these techniques suffer from the disadvantages of high costs,
sophisticated instruments, and high requirements for testing
staff possessing master professional skills and sample pretreat-
ment, resulting in the fact that they are not suitable for on-site
detection in most settings, especially in emergency cases.
Recently, novel nanomaterials have attracted great attention
and have been intensively studied in biological analysis and
detection because of their unique chemical or physical
properties.22,23 Currently, three kinds of approaches involving
nanoparticles have been developed for the detection of
pesticide exposure: (1) The first is the use of nanomaterial as
enzyme carriers for loading a large amount of enzymes to
enhance the detection signal.24 Some of these approaches
require a complex electrode modifying process or introduce
additional substrates, which make a complex detection
protocol. (2) The second is the use of nanomaterial as
peroxidase- or oxidase-like activities catalysts to catalyze the
oxidation of various substrates including 2, 2′-azino-bis(3-
ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt and
3,3,5,5-tetramethylbenzidine by enzyme-generated hydrogen
INTRODUCTION
■
P
esticides are widely used in agriculture all over the world,
whereas they cause widespread contamination of air, water,
soil, and agricultural products, eventually leading to long-term
accumulation in ecosystems including humans.1 The residues
with highly toxic substances have been found to cause serious
problems to human health even at very low concentrations.2,3
According to the World Health Organization, 1.5 billion cases
of diarrhea in children (leading to more than 3 million deaths)
are caused by contaminated food and drinking water every
year.4 The high toxicity of pesticides is ascribed to their ability
to irreversibly inhibit the activity of acetylcholinesterase
(AChE) in the central and peripheral nervous system, resulting
in the accumulation of the neurotransmitter acetylcholine in the
body, which can lead to fatal consequences.5 In order to avoid
possible harm to humans and animals, it is of great importance
to develop a highly sensitive and reliable assay for pesticide
residues to improve food safety and to protect the ecosystem.
In the past decade, a number of analytical techniques have
been devised to detect pesticides in food and water, such as
fluorescent bioprobes,6−9 colorimetric assay,10,11 chromatog-
raphy−mass spectrometry,12−14 electrochemical analysis,15−18
and enzyme-linked immunosorbent assay.19−21 Although these
methods have high selectivity and adequate sensitivity, most of
Received: August 14, 2013
Accepted: October 28, 2013
© XXXX American Chemical Society
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peroxide (H2O2) for colorimetric or fluorescence detection of
pesticides.25,26 (3) The third is the use of nanomaterial as a
direct signal source. For instance, Li’s group developed a new
approach for electrochemical quantification of organophospho-
rus pesticides using the stripping voltammetric signal of Cd2+
based on a CdTe QDs-gold nanoparticle composite.27 Gao and
colleagues reported the sensing of organophosphorus com-
pounds based on a Mn-doped ZnSe QDs−enzyme−H2O2
fluorescence quenching system.28 However, the heavy metal
ion-containing QDs may suffer from intrinsic limitations such
as potential toxicity, intrinsic blinking, and chemical insta-
bility.29 Furthermore, the release of heavy metal ions may
inhibit the activity of enzymes and is a potential environmental
hazard, which limits the application of these QDs.30 Therefore,
it is also important to develop excellent nanomaterials for
fabricating highly sensitive and selective biosensors.
Silicon quantum dots (SiQDs), as inert, nontoxic, abundant,
and low-cost nanomaterials, have been demonstrated to be
environmentally friendly photoluminescence probes and have
attracted much interest. In comparison to other QDs, SiQDs
have unique optical and electronic properties, especially
favorable biocompatibility. These advantages enable them to
play a great role in a variety of applications. To date, a few
studies have reported that the modified SiQDs may be excellent
candidates for biological imaging, but there were few studies on
the sensitive detection of various analytes associated with
enzyme-catalyzed events by using label-free SiQDs.
In this study, we report a SiQDs-based simple and highly
sensitive sensor for pesticides. It is well-known that AChE can
hydrolyze acetylcholine chloride (ACh) to choline, which can
be oxidized to betaine with the concomitant generation of
H2O2 in the presence of choline oxidase (ChOx).31 The activity
of AChE can be inhibited by pesticides.32 Our previous work
has been proved that the fluorescence of label-free SiQDs could
be effectively quenched by enzyme-generated H2O2.29 As
shown in Figure 1, a novel strategy for pesticide detection was
hence proposed. The sensing procedure is based on SiQDs
fluorescence quenching by enzyme-generated H2O2 and the
activity of AChE inhibited by pesticides. These proposed
biosensors developed a new method for biomonitoring of trace
pesticide exposures based on their inhibition effects on enzyme
activity. This new type of biosensor does not require complex
modification and enzyme immobilization, and the assay results
can be read as soon as the probe-sample incubation is
completed.
EXPERIMENTAL SECTION
■
Materials and Instrumentation. All chemicals from
commercial sources were of analytical grade and used without
further purification. AChE (from Electrophorus electricus), ChOx
(from Alcaligenes sp.), and ACh were obtained from Sigma-
Aldrich. Carbaryl, parathion, diazinon, and phorate were from
Huaerbo Chemical Reagent Co. Silicon wafers (phosphorus-
doped (p-type), 8Ω resistivity) and phosphomolybdic acid
(POM) were purchased from Sigma-Aldrich. Anhydrous
ethanol (analytical grade), hydrofluoric acid (HF), and H2O2
were purchased from Shanghai Chemical Reagent. PBS buffer
(pH 8.0, 5 mM sodium phosphate) and Milli-Q ultrapure water
(Millipore, ≥ 18 MΩ cm) were used throughout.
SiQDs synthesis was conducted on a CHI660A electro-
chemical workstation (CHI Instrument Inc., USA). UV−vis
spectra were recorded with a UV2450 spectrophotometer
(Shimadzu). The fluorescence spectra were collected on a
Hitachi F-4500 fluorescence spectrophotometer operating at
the excitation wavelength of 360 nm, with both excitation and
emission slit widths of 10 nm. The fluorescence intensities at
440 nm were measured. Transmission electron microscopy
(TEM) images were obtained by using a JEOL-1230 model.
Fourier transform infrared (FTIR) spectra were collected on a
Nicolet Nexus 670 FTIR spectrometer (Nicolet Instrument
Co., U.S.A.). High-performance liquid chromatography
(HPLC; Agilent 1200, U.S.A.) analysis was performed on a
Zorbax SB-C18 column (150 mm × 4.6 mm i.d., 4 μm). A
mixture of methanol−water (80:20 v/v) was used as the
isocratic mobile phase to deliver the sample containing carbaryl
with a flow rate of 0.8 mL/min at 25 °C. Apple, tomato, and
cucumber solutions were prepared with a juice extractor
(Philips, HR2010, 350 W) bought from a supermarket.
Synthesis of SiQDs. Photoluminescence SiQDs were
synthesized by the POM-assisted electrochemical etching of
bulk Si. Briefly, 0.015 g of POM was dissolved in 35 mL of
anhydrous ethanol, then 35 mL of HF was added under stirring.
Until the solution became transparent, a silicon wafer and
carbon rod (length of 5 cm) were immerged, where the silicon
wafer worked as the working electrode and the carbon rod
served as the reference and counter electrode. An ampero-
metric i−t curve was selected. The initial potential was about
7−10 V, and the current density was kept in the range of 4−10
mA cm−2. After etching for about 2 h, large amounts of SiQDs
formed on the surface of the silicon wafer. Without complicated
centrifugation and filtration, just ultrasonication fracturing of
the etched silicon wafer in absolute ethanol occurred, and then
SiQDs with excellent fluorescence were obtained. The prepared
SiQDs were characterized using fluorescence spectroscopy and
TEM.
Experimental Procedure for the Sensitive Assay of
Pesticides. Pesticides inhibit the activity of the AChE enzyme
and induce a decrease in the PL quenching efficiency of SiQDs
by enzyme-generated H2O2. One of the most commonly used
pesticides, carbaryl, was investigated as the representative
model pesticide. Carbaryl was determined by measuring the
decrease in quenching efficiency of the SiQDs PL in the
Figure 1. Schematic illustration of the measuring principle of the
pesticide biosensor. Inset: PL spectra of SiQDs mixtures, (a) only
SiQDs and buffer, (b) SiQDs + 2 U/mL AChE + 0.6 U/mL ChOx +
1.0 mM ACh + 8.0 × 10−5 g/L carbaryl, (c) SiQDs + 2 U/mL AChE +
0.6 U/mL ChOx + 1.0 mM ACh, and (d) SiQDs + 0.3 mM H2O2,
respectively.
B
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Figure 2. (A) Transmission electron microscopy image of SiQDs. The inset shows the high-resolution transmission electron microscopy image of
SiQDs. (B) UV−vis absorption and excitation spectra (solid line) and emission spectra (dot line) of SiQDs. The inset shows the photo of SiQDs
illuminated by UV light of 365 nm.
presence of the AChE and ChOx. AChE (2 U/mL) was first
incubated with various concentrations of carbaryl in 50 μL of
PBS (5 mM, pH 8.0) for 10 min at room temperature. The
resultant solution was then added to the assay solution
prepared by suspending the SiQDs in 450 μL of PBS (5
mM, pH 8.0) which contained ChOx (0.6 U/mL) and ACh (1
mM). The mixed solution was incubated for another 15 min in
the dark at 40 °C. The PL intensity of SiQDs at 440 nm was
collected in the final reaction solution.
Analysis of Carbaryl Residues in Food Sample. Apple,
tomato, and cucumber were chosen as the sample matrix to
evaluate the carbaryl residue levels in the real application tests
of this pesticide assay. A standard carbaryl solution (0.1 g/L)
was sprayed onto skins of the food samples by an atomizer, and
the carbaryl residues in aliquots of the samples were collected
every other day over the course of 10 days. The samples were
first chopped, and the edible parts of the samples were crushed
into a homogenate by a juice extractor. A total of 25 g of each
homogenate was mixed with 50 mL of acetonitrile, and the
resulting mixture was filtered through a 0.22 μm membrane to
remove the insoluble materials. The obtained filtrate was dried
on a water bath, and the remaining solid substance was mixed
with methanol to a final volume of 5 mL. Subsequently, 30 μL
of the final carbaryl residue solution was added to 1 mL of the
SiQDs, AChE, ChOx, and ACh mixture solution (SiQDs−
AChE−ChOx−ACh) and incubated in a water bath at 40 °C
for 15 min. Then, the mixture was taken out from the water
bath and allowed to cool to room temperature for 5 min for PL
measurements at an excitation wavelength of 360 nm.
coupled H−Si−Si−H stretch bonds occurred at 2100 cm−1.35
The PL quantum yield of SiQDs was up to ∼9.6% according to
the Williams method36 (the detailed process shown in the
Supporting Information). Compared with other reported
methods, the obtained SiQDs have very high quantum
yields.37,38 Upon further investigation, we found that the PL
intensity of SiQDs was not affected by temperature (Figure
S2A, Supporting Information) and reached a maximum when
the pH of SiQDs in solution was 7.5−8.0 (Figure S2B,
Supporting Information). Moreover, the SiQDs diluted with
PBS (pH 8.0) possessed excellent photostability over 6 h in air
under ambient conditions without any protection (Figure S2C,
Supporting Information).
Quenching Effect of H2O2 on the PL Emission of
Label-Free SiQDs. The proposed sensing strategy of
pesticides is based on label-free SiQDs and the enzyme
reaction of ACh. The basic principle of the SiQDs-based
pesticide sensor includes the hydrolysis of ACh catalyzed by
AChE, the oxidation of choline in the presence of ChOx, the
inhibition of enzyme activity by pesticides, and PL quenching of
SiQDs by the enzyme-generated H2O2.
AChE
ACh + H2O ⎯⎯⎯⎯⎯→ Choline
(1)
ChOx
Choline + O2 ⎯⎯⎯⎯→⎯ H2O2 + Betaine
(2)
To determine the concentration of pesticides, the correlations
of the inhibition effects of pesticides, the enzyme-generated
H2O2, and the PL quenching effect of SiQDs should be
established. Herein, we first investigated the influence of the
H2O2 or AChE−ChOx−ACh in the absence and presence of
carbaryl on the PL emission of the SiQDs. As can be seen from
Figure 1, the PL intensity of the label-free SiQDs solution
evidently decreased after incubation with H2O2 (curve d) or
AChE−ChOx−ACh (curve c). While the label-free SiQDs were
individually incubated with ChOx, AChE, ChOx and AChE,
ACh, or carbaryl under the same conditions, almost no change
was observed in SiQDs PL (Figure S3, Supporting
Information). In addition, when the AChE−ChOx−ACh was
pretreated with carbaryl before it was incubated with the label-
free SiQDs, the SiQDs PL decrease was effectively prevented.
In order to determine the effect of oxygen dissolved, time-
dependent PL spectra of SiQDs−AChE−ChOx system in the
saturated oxygen atmosphere were investigated (Figure S4,
Supporting Information). The results indicated that the
SiQDs−AChE−ChOx system possessed excellent photostabil-
ity over 60 min in the saturated oxygen atmosphere and implied
that the oxygen dissolved in the SiQDs−AChE−ChOx system
RESULTS AND DISCUSSION
■
Characterization of the SiQDs. The morphology and
optical properties of SiQDs were characterized, and the results
are shown in Figure 2. TEM (Figure 2A) and the high
resolution transmission electron microscopy (HRTEM; inset of
Figure 2A) images show that the SiQDs were mostly spherical
dots, and the size is uniform, with an average diameter of about
5 nm. The absorption, excitation, and emission spectra of
SiQDs in aqueous solution are presented in Figure 2B. The
absorbance below 290 nm is a characteristic absorbance of
SiQDs.33 A narrow emission spectrum range from 400 to 500
nm was observed with the SiQDs solution, which illustrates that
the size of the SiQDs is uniform.34 As shown in the inset of
Figure 2B, the SiQDs emitted bright blue light under UV light
excitation. The prepared SiQDs are terminated with Si−H
bonds, as revealed by FTIR measurement (Figure S1,
Supporting Information). The strong stretching modes of
monohydrides Si−H bonds occurred at around 900 cm−1, and
C
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Figure 3. SiQDs PL intensity vs ACh concentration. The concentrations of AChE and ChOx are 2 U/mL and 2 U/mL (A), 2 U/mL and 0.6 U/mL
(B), 5 U/mL and 0.3 U/mL (C), and 0.3 U/mL and 2 U/mL (D), respectively.
has no influence of the SiQDs PL. Figure S5 (Supporting
Information) showed the PL intensities of SiQDs solution after
treatment with different amounts of H2O2. It was observed that
the PL intensity of SiQDs decreased gradually with the
increasing of H2O2. These facts assuredly testified to the
quenching effect of the enzyme-generated H2O2 on SiQDs PL
and the inhibition effect of pesticides on the enzyme activity.
The PL quenching mechanism of SiQDs by H2O2 (from
catalytic oxidation of choline generated from the reaction of
ACh with AChE) can be explained as follows: Since H2O2
contains the active oxygen species, it can diffuse across the
surface of the SiQDs and capture the electrons at the
conduction bands of SiQDs, and subsequently the radiative
recombination of the photoinduced electrons and holes is
inhibited.39 Therefore, H2O2 was capable of capturing the
electrons of SiQDs as well as the quenching of the SiQDs PL.34
The quenching process was also supported by TEM analysis
(Figure S6, Supporting Information). The SiQDs remained
well dispersed and highly uniform in size after the treatment of
AChE−ChOx−ACh. But, the lattice fringe became blurred (the
HRTEM image inset in Figure S6, Supporting Information),
suggesting that the reaction of H2O2 and SiQDs occurred.
Upon the above observations, we deduce that the inhibition
efficiency of pesticides on the activity of the enzyme could be
correlated with the quenching extent of the SiQDs PL. By
measuring the SiQDs PL of the carefully designed test system,
the quantification of pesticides can be achieved.
significant differences between the results in the presence
(Figure S7A and S7C, Supporting Information) and absence
(Figure S7B and S7D, Supporting Information) of SiQDs,
indicating that the SiQDs have no effect on enzyme activity.
Optimization of the Detection Conditions. To
sensitively detect pesticides, figuring out the influences of the
incubation time, temperature, and pH value on the SiQDs PL is
necessary. As shown in Figure S8A (Supporting Information),
the value of the quenching efficiency ((I0 − I)/I0, I0 and I refer
to the PL intensity of SiQDs−AChE−ChOx system in the
absence and presence of ACh) reached a maximum when the
temperature reached 40 °C. While verifying the pH value of the
system in the presence of 1.0 mM ACh (Figure S8B,
Supporting Information), the quenching efficiency reached a
maximum at pH 8.0. Figure S8C (Supporting Information)
shows that the quenching efficiency sustained a stable value
when the incubation time reached 15 min. On the basis of
those observations, the analyte systems were incubated at 40 °C
for 15 min under a pH of 8.0 before fluorescence measurement
in further experiments.
The concentrations of AChE and ChOx would have
influences on the sensitivity of the proposed sensing strategy
for pesticide detection, because the concentrations of AChE
and ChOx have an effect on the enzyme generated H2O2 in the
two-enzyme coupled reaction system and consequently affect
the SiQDs PL intensity. This conjecture was experimentally
confirmed by the results presented in Figure S9 (Supporting
Information). Performing linear regression analysis between the
SiQDs PL intensity and the ACh concentration presented in
Figure S9(A−D) (Supporting Information), four linear
equations were obtained, which could be expressed as (A) Y
= −0.1712X + 0.9927, (B) Y = −0.3016X + 0.9836, (C) Y =
−0.1098X + 0.9849, and (D) Y = −0.2100X + 0.9801,
respectively, where Y refers to the PL intensity and X represents
the concentrations of ACh. Figure 3 shows linear regression
results, the PL intensity of the SiQDs−AChE−ChOx system
decreased with the increasing concentration of ACh in all tested
Influence of SiQDs on Enzyme Activity. It is important
to evaluate the influence of the label-free SiQDs on the activity
of the used enzyme for pesticide detection following the
proposed sensing strategy. The Ministry of Health Determi-
nation Standard of the AChE and ChOx (WS/T66-1996) has
been selected for monitoring the enzyme activity changes in the
presence and absence of SiQDs.40 The absorbance spectra and
color changes of variable concentrations of ACh upon the
interaction with AChE, ChOx, and alkaline hydroxylamine are
shown in Figure S7 (Supporting Information). There are no
D
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cases. The absolute value of the slope (|k|) of the four lines is
ordered as |kB| > |kD| > |kA| > |kC|, which indicates that the
highest quenching effect was reached when the concentrations
of AChE and ChOx were 2 U/mL and 0.6 U/mL, respectively.
Therefore, we use 2 U/mL AChE and 0.6 U/mL ChOx in
further experiments.
Therefore, in the present study, an ACh concentration of 1.0
mM was chosen for pesticide determination.
(I0 − I )
10
K10
=
(3)
10
(K10without − K10with
)
Similar to the previous reports,28,41 the absolute quenching
rate is related to the Stern−Volmer constant. From Figure 5A,
the linear Stern−Volmer plots (PL intensity vs incubation
time) are obtained with K10 values (which were calculated as
the slope of the plot of the PL intensity change vs reaction time
for the first seven data points (the first 10 min)). K10 values
were calculated via eq 3, where I0 and I10 are the PL intensity
recorded at 440 nm before and after the addition of ACh (first
10 min), respectively. The inhibition efficiency (IE) values were
calculated via eq 4, where K10without and K10with are the absolute
quenching rates without inhibition and with inhibition at a
certain concentration of pesticides, respectively. On the basis of
the inhibition efficiency, the optimized concentration of ACh
was tested as well. As shown in Figure 4, the inhibition
IE =
× 100
K10without
(4)
Sensitive Assay for Pesticides. Figure 5A shows the time-
dependent PL intensity of SiQDs upon treatment of the
SiQDs−AChE−ChOx system in the presence of 1.0 mM ACh
with variable concentrations of carbaryl. As the time was
prolonged, the quenching effect of the QDs was reinforced.
The result indicated that the SiQDs had been quenched by
H2O2 which generated during the AChE and ChOx catalytic
reactions. Figure 5B shows the relationship between inhibition
efficiency and the carbaryl concentration, which inferred that
the inhibition efficiency was linearly dependent on the
logarithm of the carbaryl concentration in the concentration
range of 7.49 × 10−9 g/L to 7.49 × 10−4 g/L, and the
corresponding linear function is IE (inhibition efficiency, %) =
15.4552 log[carbaryl] (g/L) + 130.8024 with a correlation
coeffcient of R2 = 0.9903. The limit of detection (LOD; defined
as the concentration of inhibitor required to achieve 5%
inhibition43) was 7.25 × 10−9 g/L, which is much lower than
the maximum residue limits (MRLs) as reported in the
European Union pesticides database (MRL is 0.05 ppm for
carbaryl)44 as well as that of the United States (MRL is 0.02
ppm for carbaryl).45
To demonstrate that this assay method was effective not only
for carbaryl but also for other pesticides, we investigated the
sensitivity of the sensing platform for three other conventional
pesticides: parathion, diazinon, and phorate. The experimental
procedures were the same as that for carbaryl. Figure S10A,C,E
show incubation time dependence of the PL intensity of the
SiQDs−AChE−ChOx system vs variable concentrations of
diazinon (A), parathion (C). and phorate (E). They had a
similar trend to that of carbaryl. The lowest detectable
concentrations for parathion, diazinon, and phorate are 3.25
× 10−8, 6.76 × 10−8, and 1.9 × 10−7 g/L, respectively, which is
much lower than those previously reported (Table S1).
Inhibition Efficiencies of the Pesticides. As different
pesticides would have different inhibition efficiencies with
respect to AChE, the inhibition efficiency of four pesticides at
the concentration of 8.0 × 10−7 g/L was also investigated and is
Figure 4. Inhibition efficiencies of carbaryl on enzyme vs ACh
concentrations. The AChE, ChOx, and carbaryl concentrations are 2
U/mL, 0.6 U/mL, and 8.0 × 10−7 g/L, respectively.
efficiency increased with the increasing concentration of ACh
from 0 to 1.0 mM, and then the response shows a slight
decrease when the concentration of ACh is higher than 1.0
mM, which could be attributed to the phenomenon known as
substrate inhibition.42 When the ACh concentration was higher,
the high concentration of enzymes was not conducive to the
quenching effect of H2O2, because the excess ACh covered on
the surface of SiQDs would inhibit the quenching effect.
Figure 5. (A) Incubation time dependence of the PL intensity of the SiQDs−AChE−ChOx system in the presence of 1.0 mM ACh and a variable
concentration of carbaryl. Carbaryl concentration (a−g): 0, 7.49 × 10−9 g/L, 7.49 × 10−8 g/L, 7.49 × 10−7 g/L, 7.49 × 10−6 g/L, 7.49 × 10−5 g/L,
and 7.49 × 10−4 g/L. (B) Inhibition efficiency vs the logarithm of carbaryl concentration. All measurements were performed in PBS, pH = 8.0. The
concentrations of AChE and ChOx are 2 U/mL and 0.6 U/mL, respectively.
E
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shown in Figure S7 (Supporting Information). It was observed
that the IE values of carbaryl, diazinon, and parathion were
considerably higher than that of phorate (12.48%), which were
32.47%, 30.85%, and 26.48%, respectively. The differences in
the inhibition efficiency may result from the disparities in the
molecular characteristics of the pesticides and agreed with the
quantum chemistry simulations (Gaussian 03).46 The changes
in the conformation of AChE on the surface of SiQDs may also
be responsible to these observations. Further studies on how
the molecular characteristics of pesticides and conformational
changes affect the inhibition efficiency are demanded in future
work.
Anti-Interference Capability of the Sensor. It is
essential to estimate the anti-interference capability of the
sensor from the coexisting species in the analyte. Because this
work is aimed at the development of a new sensing strategy for
detecting pesticide residues, the common existing substances in
food samples, such as glucide, metal ions, organic acids, and
amine acids, were studied as the controls. As shown in Figure 6,
Figure 6. Effects of possible interference species in analytes (1.0 ×
10−3 g/L) on the inhibition efficiency of carbaryl (8.0 × 10−7 g/L) in
the SiQDs−AChE−ChOx system.
Figure 7. Analytical results of carbaryl in apple (A), tomato (B), and
cucumber (C) samples obtained by using the present SiQDs−AChE−
ChOx based method (black) and by using HPLC (red). All
measurements were performed in PBS, pH = 8.0, thermostated at
40 °C.
glucose, sucrose, fructose, galactose, and glycerol exhibited little
influence on carbaryl detection even at concentrations 103
times higher than that of carbaryl. Typical metal ions, organic
acids, and amine acids almost showed no interference in
carbaryl determination. These results clearly demonstrate that
the SiQDs based PL sensor is of high specificity for pesticide
detection and has a strong capability to resist interference.
Determination of Carbaryl Residues in the Food
Samples. The excellent specificity and high sensitivity of the
sensor suggest that the developed method might be directly
applied for detecting pesticide residues in real samples.
Therefore, we further investigated whether the sensing strategy
described here could be utilized to monitor the residues of
pesticides in fruit and vegetable samples such as apples,
tomatoes, and cucumbers. The carbaryl residues in aliquots of
the food samples were collected every other day over the course
of 10 days. The concentration of carbaryl residue was
determined using a novel sensing strtegy based on a SiQDs−
AChE−ChOx−ACh system and was also analyzed with HPLC
to test the accuracy of this method. The carbaryl concentrations
in food samples detected by the proposed assay and HPLC are
shown in Figure 7. The results obtained by using this assay
coincided well with those obtained by using HPLC, indicating
that the proposed method can be successfully applied for
detecting pesticide residues in real samples.
CONCLUSIONS
■
In conclusion, we have developed a novel label-free SiQDs-
based sensor for the detection of pesticides. The two-enzyme
(AChE and ChOx) coupled reaction system is involved in this
new developed sensing strategy. It has been experimentally
demonstrated that the label-free SiQDs have no effect on the
enzyme activity, and the enzyme generated H2O2 can effectively
quench the PL of SiQDs. As the PL of SiQDs is very sensitive
to H2O2, the measurements of SiQDs PL in the designed
system could be used to evaluate the amount of the enzyme
generated H2O2, which reflects the activity level of the enzymes.
And it is found that the inhibitory effect of pesticides on the
enzyme activity (termed the inhibition efficiency and evaluated
by measuring the SiQDs PL) was linearly dependent on the
logarithm of the pesticide concentration. The lowest detectable
carbaryl concentration in this newly developed sensing strategy
is much lower than the maximum residue limits of the
European Union and U.S. Department of Agriculture, and the
accuracy of this assay in real sample analysis is comparable to
the traditional HPLC method, which indicates that the sensing
strategy is of prospective application for pesticide residue
detection in foodstuffs. Because this assay has the significant
advantage of being simple, rapid, highly sensitive, and capable
of anti-interference in pesticide detection, we believe this assay
F
dx.doi.org/10.1021/ac403257p | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
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addition, the research provides a new approach to further
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ASSOCIATED CONTENT
* Supporting Information
Additional information as noted in the text. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
(25) Burmeister, J. J.; Pomerleau, F.; Huettl, P.; Gash, C. R.; Werner,
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +86-731-88865515. Fax: +86-731-88865515. E-mail:
zhangyy@hunnu.edu.cn.
■
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Notes
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The authors declare no competing financial interest.
(31) Wang, F.; Liu, X.; Lu, C.-H.; Willner, I. ACS Nano 2013, 7,
7278−7286.
ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science
Foundation of China (21275051, 21375037), Scientific
Research Fund of Hunan Provincial Education Department
(12A084) and Science and Technology Department
(13JJ2020), and Aid Program for Science and Technology
Innovative Research Team in Higher Educational Institutions
of Hunan Province.
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