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Article
Quantum dot-based lateral flow immunoassay for
detection of neonicotinoid residues in tea leaves
Shuangjie Wang, Ying Liu, Shasha Jiao, Ying Zhao, Yirong Guo, Mengcen Wang, and Guonian Zhu
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03981 • Publication Date (Web): 27 Oct 2017
Downloaded from http://pubs.acs.org on October 27, 2017
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Journal of Agricultural and Food Chemistry is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
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Page 1 of 34
Journal of Agricultural and Food Chemistry
1
2
3
Quantum dot-based lateral flow immunoassay
for detection of neonicotinoid residues in tea
leaves
4
5
Shuangjie Wang1, Ying Liu1, Shasha Jiao1, Ying Zhao1, Yirong Guo1,
6
Mengcen Wang1†, Guonian Zhu1
7
8
1
Institute of Pesticide and Environmental Toxicology, Zhejiang
University, 310058 Hangzhou, China
9
10
†Address for Correspondence:
11
Mengcen Wang, Ph.D.
12
Institute of Pesticide and Environmental Toxicology, Zhejiang University
13
E-mail: [email protected]
14
Phone & Fax: +86-571-88982517
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Abstract
16
Neonicotinoid insecticides are commonly used for pest control on tea
17
plantations due to their broad-spectrum activity. However, neonicotinoid
18
residues released from tea leaves into tea infusions pose a dietary risk to
19
consumers. Therefore, a rapid, sensitive and reliable on-site detection
20
method for neonicotinoids is needed. We developed a quantum dot-based
21
fluorescent lateral flow immunochromatographic strip (LFICS) combined
22
with a broad-specific antibody for detection of typical neonicotinoids
23
(imidacloprid, imidaclothiz, and clothianidin), with sensitivities (IC50, 50%
24
inhibitory concentration) of 0.104–0.33 ng/mL and visual detection limits
25
of 0.5–1 ng/mL. The strip assay could be completed in less than 30
26
minutes. Using the LFICS to analyze spiked tea samples (green tea, black
27
tea, and oolong tea), the average recovery of the three neonicotinoids
28
ranged between 71% and 111%, with coefficients of variation below 12%.
29
The results from the LFICS tests for field samples were consistent with
30
results from ultra-performance liquid chromatography-tandem mass
31
spectrometry. The newly-developed strip is a useful tool for the on-site
32
detection of neonicotinoid residues in tea.
33
34
Keywords: lateral flow immunoassay; quantum dot; neonicotinoids; tea
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36
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Page 3 of 34
Journal of Agricultural and Food Chemistry
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Introduction
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Teas can be classified into three types (green tea, oolong tea and
39
black tea) based on fermentation processing1. Tea plants are attacked by
40
many sucking insects, and insecticides are commonly used for their
41
control. Among the insecticides used, neonicotinoids are effective for
42
controlling whiteflies, aphids, and leaf hoppers. Neonicotinoids have the
43
advantages of a broad host spectrum, long residual activity and unique
44
modes of action. They have become the most widely used insecticides in
45
the world 2. Neonicotinoids are relatively polar compounds and are easily
46
leached from dry tea or the surface of treated tea into drinkable tea
47
infusions. This creates a risk of human exposure to these pesticide
48
residues 3. Hence, it is necessary to monitor neonicotinoid residues in teas
49
to increase tea quality and safety.
50
Various instrumental analytical approaches have been used to detect
51
neonicotinoids. These include high-performance liquid chromatography
52
coupled
53
chromatography tandem mass spectrometry (LC-MS/MS)5, and ultra
54
performance
55
(UPLC-MS/MS)6, 7. All of these methods are acceptably sensitive,
56
accurate and selective, but the processes are complex, and the necessary
57
equipment is expensive. Therefore, a portable, sensitive, rapid and
58
easy-to-use method, which can also be used outside the laboratory, was
with
a
liquid
diode
array
detector
(HPLC-DAD)4,
chromatography-tandem
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mass
liquid
spectrometry
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Page 4 of 34
developed for neonicotinoid detection.
60
Immunoassays, based on antibody-antigen interactions, have been
61
widely applied in disease diagnosis and biochemistry. Immunoassays
62
benefit from the specific recognition of antigen by an antibody, which
63
reduces the onerous procedures of sample pretreatment. Enzyme-linked
64
immunosorbent assay (ELISA) is a common immunoassay for residue
65
analyses of neonicotinoids
66
and cannot be used for rapid on-site screening. In contrast, one-step
67
lateral flow immunochromatographic strips (LFICSs) can reduce some of
68
the ELISA difficulties 10.
69
8, 9
. However, ELISA requires multiple steps
Colored nanoparticles and luminescence materials have been used as
11,
12
70
detection probes in LFICS
. Among these, colloidal gold,
71
characterized by its tunable optical properties and stability under liquid
72
and dry conditions, is a common probe used in LFICS. Several studies
73
have used nanogold-based LFICS for the detection of neonicotinoids. A
74
nanogold-based immunostrip was developed for the simultaneous
75
detection of imidacloprid and thiamethoxam, and the visual detection
76
limits in the assay buffer were 0.5 and 2 ng/mL, respectively
77
nanogold-based signal amplified immunochromatographic assay was
78
developed for semi-quantitative detection of imidacloprid
79
studies describing the use of other nanoparticle-labelled strips for the
80
detection of neonicotinoids have not been reported.
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. A
14
. However,
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Journal of Agricultural and Food Chemistry
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Quantum dot (QD) is a new type of fluorescent nanoparticle
82
semiconductor material. It is characterized by its broad adsorption,
83
narrow photoluminescence spectra, size-tunable emission, strong
84
luminescence and high photostability
85
functional groups, such as carboxyl, to achieve water solubility and
86
biocompatibility. Their unique properties make them suitable for a wide
87
array of biotechnological and bio-analytical studies
88
LFICS consumed less immunoreagents and was more sensitive than the
89
colloidal gold-based LFICS
90
immunostrips. Thus far, QDs have been used as probes in
91
microwell-based fluorescent-linked immunosorbent assays for the
92
detection of neonicotinoids such as clothianidin and thiacloprid
93
imidaclothiz
94
detection, and no studies of QD-based LFICS for neonicotinoid detection
95
have been published. In this study, we used QD as the label to develop a
96
rapid, sensitive, portable fluorescent LFICS for detecting three
97
neonicotinoids (imidacloprid, imidaclothiz, and clothianidin) in tea
98
samples.
23
15
. QDs can conjugate with
16-18
. The QD-based
19-21
. Therefore, QD is a promising label for
22
and
. There are few reports of QD-based LFICS for pesticide
99
100
Material and methods
101
Reagents and materials
102
Standards of eight neonicotinoids (imidacloprid (99.0%), dinotefuran
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(97.5%), nitenpyram (99.0%), acetamiprid (99.0%), imidaclothiz (99.0%),
104
thiacloprid (98.0%), thiamethoxam (99.0%), clothianidin (99.5%), were
105
purchased from Dr. Ehrenstorfer (Augsburg, Germany). The carboxylic
106
group-modified CdSe/ZnS core-shell QDs (emission at 605 ± 5 nm) were
107
provided by Jiayuan Quantum Dots Co., Ltd. (Wuhan, China). A
108
broadly-specific monoclonal antibody (mAb) against imidacloprid and its
109
analogues was previously prepared in our laboratory, as well as the
110
corresponding coating antigen. The 1-ethyl-3-(3-dimethylaminopropyl)
111
carbodiimide hydrochloride (EDC) Tween-20, bovine serum albumin
112
(BSA), polyvinyl pyrrolidone (PVP) and sucrose were provided by
113
Aladdin
114
secondary amine, PSA) was obtained from Agela Technologies (Tianjin,
115
China). Polyvinyl polypyrrolidone (PVPP) was obtained from Sigma
116
(Steinheim, Germany). All other inorganic chemicals and organic
117
solvents were of analytical reagent grade or better. Purified water was
118
obtained using a Milli-Q water purification system (Millipore, Bedford,
119
MA, USA). Glass-fiber membrane CFCP203000 was used for loading
120
conjugate and the absorption membrane CFSP223000 were from
121
Millipore. The nitrocellulose (NC) membranes were purchased from
122
different companies, including Sartorius-CN-140 (Gottingen, Germany),
123
Millipore HiFlow-135 and HiFlow-180 (Billerica, USA).
124
Preparation of QD-mAb conjugates
(Shanghai,
China).
N-propyl-ethylenediamine
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(primary
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Journal of Agricultural and Food Chemistry
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The conjugation strategy for the preparation of QD-based antibody
126
was modified from a previous report 22. A quantity of carboxyl-modified
127
QDs (25 µL, 8 µM in 50 mM borate buffer, pH 9.0) was mixed with
128
borate buffer (10 mM, pH 8.0) under magnetic stirring. Then, the
129
antibodies (1.64 – 24.5 µL, 9.17 mg/mL) and EDC (7.7 µL, 10 mg/mL)
130
were added into the previous solution. The mixture (200 µL) was
131
incubated for 120 min at 4 °C in darkness with stirring at 320 rpm. This
132
was followed by centrifugation (5000 rpm, 10 min) by a 5-mL
133
ultra-filtration concentrator (MWCO 3K, Millipore). The supernatant was
134
then removed and the conjugate was resuspended with borate buffer (10
135
mM, pH 7.5) containing 1% BSA, 0.05% PVP and 1% sucrose. The final
136
conjugate solution was stored at 4°C.
137
Assemblage of the one-step strips
138
A one-sided adhesive polyvinyl chloride (PVC) sheet was used as a
139
support for the strip composition. The absorption membrane and the glass
140
fiber membrane were pasted on the sheet, overcrossing 2 mm with the
141
two ends of the NC membrane. These composites were stored in a
142
desiccator at 4 °C before use.
143
Preparation of QD-based LFICS
144
Coating antigen and goat anti-mouse antibody were immobilized
145
onto the NC membrane as the test and control lines, respectively. The
146
membrane was dried at 37°C for 2 hr. The obtained composite was cut
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into individual strips and stored in a desiccator at 4°C before use.
148
Strip assays for imidacloprid
149
The QD-mAb conjugate was diluted (400X) by borate buffer before
150
using. Standard or sample solution (25 µL) and the QD-mAb conjugate
151
(25 µL) were mixed and added to wells of the 96-well microtiter plate.
152
Then, the strip was inserted into the well of 96-well plate. After 25 min,
153
the visible signal was observed under 365nm UV excitation and the
154
fluorescence intensity was recorded by a fluorescent reader (365 nm
155
excitation, 610 nm emission), as shown in Fig. 1.
156
To offset the heterogeneity of the strips, the fluorescence intensity
157
ratio of T-line to C-line (T/C) was used for quantitative analysis and this
158
minimized environmental factors potentially affecting fluorescence
159
intensity. Standard curves were obtained by plotting the fluorescence
160
intensity ratio of the T-line to the C-line (T/C) (as Y-axis) against the
161
analyte concentration (X), and they were fitted into a four-parameter
162
logarithmic equation.
163
Y = A2 + [(A1- A2) / 1 + (X / X0)p], where A1 is the maximum value of
164
T/C in the logarithmic equation, while A2 is the minimum value; X0 is
165
equal to IC50 (50% inhibitory concentration), taken as the assay sensitivity,
166
and p indicates the slope of the curve at IC50. The linear working range
167
was represented by IC20–IC80.
168
Cross-reactivity
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Cross-reactivity (CR) was used to express the selectivity of the strip
170
assay.
171
neonicotinoids (dinotefuran, nitenpyram, acetamiprid, imidaclothiz,
172
thiacloprid, thiamethoxam, and clothianidin) were all tested by the strips.
173
CR values were calculated as follows:
174
175
The
standard
solutions
of imidacloprid
and
the
other
CR % = (IC50 of imidacloprid / IC50 of the other neonicotinoids) × 100
Tea matrix effect on LFICS
176
Neonicotinoids have good water solubility and tea is commonly
177
brewed with boiling water. Thus, each sample (1 g) of tea was extracted
178
with 10 mL of boiling water. After 30 min, the extracted tea infusions
179
were diluted with different volumes of borate buffer. Matrix effects were
180
determined by comparing standard curves in the matrix extracts with the
181
curve prepared using matrix-free borate buffer.
182
Recovery tests for tea samples
183
184
Neonicotinoid-free tea samples confirmed by UPLC-MS/MS were
used as blank samples for recovery tests.
185
Blank tea samples (1 g of dried black tea, dried green tea, or oolong
186
tea) were spiked with imidacloprid at 0.04-320 mg/kg and left standing
187
for 30 min. Then, tea samples were extracted with 10 mL of boiling water
188
for 30 min. The supernatant was then diluted with borate buffer (10 mM,
189
pH 7.5) and used for LFICS analysis. Each analysis was performed in
190
four replicates and continued for 4 d. Then, the recovery (the calculation
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191
formula listed in the supporting information) and coefficient of variability
192
(CV) were calculated.
193
Analysis of authentic samples
194
Tea samples (2.0 g) collected from different production areas (S1-S8)
195
were placed into 50-mL centrifuge tubes and 10 mL of boiling water was
196
added. The samples were incubated for 30 min. Two methods were used
197
to study neonicotinoid levels in the samples. For the strip test, the
198
supernatant was diluted with borate buffer (10 mM, pH 7.5) and then it
199
was ready for LFICS analysis. UPLC-MS/MS was also used for testing.
200
The sample-pretreatment method was modified from previous studies
201
3, 6,
24
. The incubated solution was extracted by vigorous shaking for 30 min
202
with acetonitrile (20 mL). Then, NaCl (5 g) and MgSO4 (5 g) were added
203
and the mixture was vigorously shaken for 1 min. Then, the mixture was
204
centrifuged (6000 rpm, 8 min) and the supernatant was transferred to a
205
100-mL flat-bottomed flask. The extraction was then concentrated using a
206
rotatory evaporator and dried by nitrogen gas at 40 °C. The residue was
207
dissolved in 2 mL of methanol. PSA (0.1 g) and PVPP (0.3 g) were added
208
to the residue solution, followed by vortexing for 2 min, and
209
centrifugation (6000 rpm, 5 min). The supernatant was filtrated through
210
microporous film (0.22 µm) before undergoing UPLC-MS/MS.
211
UPLC-MS/MS analysis and validation
212
The authentic samples were analyzed by UPLC-MS/MS. The UPLC
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Journal of Agricultural and Food Chemistry
213
system consisted of an Acquity ultra-performance liquid chromatograph
214
(Waters, Milford, MA). Chromatographic separations of neonicotinoids
215
were performed on a UPLC HSS C18 SB Column (1.8 µm, 2.1 × 100 mm
216
i.d. Acquity). The mobile phase consisted of 60% solvent A (0.1% of
217
formic acid in water, v/v) and 40% solvent B (acetonitrile). A subsequent
218
equilibration time (10 min) was performed before injection. The flow rate
219
was 0.3 mL/min, the injection volume was 10 µL, and the column and
220
sample temperatures were maintained at 40 °C and 8 °C, respectively.
221
The MS/MS analysis was performed by Applied Biosystems Triple
222
Quad 5500 (Foster City, CA, USA) in electrospray positive-ion multiple
223
reaction modes. The parameters of m/z and collision energy of precursor
224
ions and quantitative product ions from neonicotinoids
225
Table S1. Source–dependent parameters were as follows: ion spray
226
voltage, 5500 V; curtain gas, 20 psi; ion source temperature, 200 °C;
227
atomization air pressure, 20 psi; auxiliary gas, 20 psi; collision-activated
228
dissociation, 4 V. The AB Sciex Analyst 1.6 software (Applied
229
Biosystems) was used for data acquisition and evaluation.
24
are shown in
230
231
Results and discussion
232
The lateral flow immunoassay was based on a competitive format
233
(Scheme 1). The target pesticide in the samples and the antigen coated on
234
the test line compete for binding to the antibody conjugated with QDs. In
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the absence of pesticides in the sample, some of the Ab-QDs conjugates
236
will bind to the antigen on the test line, and the remaining conjugates will
237
bind to goat anti-mouse antibody on the control line, expressed by the
238
equalization of the fluorescence intensity between the T-line and C-line.
239
In the case of excess pesticides in a sample, the Ab-QDs combined with
240
the pesticides, and there were no Ab-QDs conjugates able to bind to the
241
T-line; the fluorescence intensity of the T-line therefore decreased.
242
Molar ratio of quantum dot to antibody
243
244
Under UV 365 nm excitation, the maximum emission wavelength of
the QDs was displayed (Fig. S1).
245
Theoretically, the free antigen in the sample solution should
246
completely occupy the binding site of antibody coupled with QDs, so that
247
the antigen immobilized on the T-line could not bind with the conjugate
248
again. However, there was often excessive coupling-antibody, which led
249
to the QD-Ab conjugate’s binding with both the free analyte and the
250
immobilized antigen. This phenomenon would decrease the sensitivity.
251
Therefore, it is important to optimize the molar ratio of the QD to the
252
antibody.
253
Foubert proposed that each QD can conjugate with 2–10
254
immunoglobulins 25. Hence, the effect of different molar ratios of QD to
255
the mAb (1:1, 1:5, 1:10, and 1:15) on the assay’s performance was tested.
256
Among these, the 1:1 group gave no fluorescence signal, and the 1:10 and
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Journal of Agricultural and Food Chemistry
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1:15 groups had lower sensitivities (Fig. 2). Thus, the 1:5 group was the
258
optimal molar ratio. The results were consistent with a previous study
259
showing that a higher molar ratio of antibody (IgG) to QD could decrease
260
assay sensitivity because it was difficult to completely block antibodies
261
by the analytes 18.
262
Optimization of experimental parameters
263
The analytical performance of LFICS can be affected by the
264
properties of the materials used to fabricate the device, particularly the
265
working membrane. Three types of NC membranes frequently used in
266
LFICS, including Millipore HiFlow-180, Millipore HiFlow-135, and
267
Satorius CN-140, were used to evaluate the sensitivity and fluorescent
268
intensity of LFICSs. All tested membranes showed an equilibrium of
269
intensity between the T-line and C-line. Sartorius CN-140 achieved the
270
desired fluorescent intensity and sensitivity (IC50) and was determined to
271
be the optimal nitrocellulose membrane (Fig. 3).
272
The concentrations of the coating antigen (0.1 to 1 mg/mL) in the test
273
line and goat anti-mouse antibody (0.01 to 0.5 mg/mL) in the control line
274
were adjusted using a matrix approach. If the T-line intensity is much
275
stronger than that of C-line, it will be judged as a false-negative and
276
reduce the sensitivity. On the contrary, it will be judged as a false-positive.
277
We found that 1 mg/mL of the coating antigen and 0.025 mg/mL of the
278
secondary antibody provided the optimal working concentrations.
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279
To prevent the non-specific binding in the assay, BSA was used to
280
block the leftover spaces over the solid surface after immobilization of a
281
captured biomolecule. Based on the additives used in previous studies
282
26,
27
, the glass-fiber was pretreated with PBST containing 0.25% BSA, 0.25%
283
PVP and 5% sucrose to provide good dispersion of coupling-QDs during
284
the strip tests.
285
To establish the optimal reaction system for good sensitivity and
286
good signal intensity for both the T-line and C-line, 10 mM borate buffer
287
at different pH levels of 7.0, 7.5, 8.0, and 8.5 were evaluated. There was
288
no obvious difference in sensitivity and T /C among these groups (Fig. 4).
289
Similar to other reports
290
condition.
291
Determination of imidacloprid by LFICS
20, 22, 28
, pH 7.5 was chosen as the working
292
Under the optimal conditions, a series of known concentrations
293
(0.024 to 100 ng/mL) of imidacloprid were prepared with borate buffer
294
(10 mM, pH 7.5) to produce a standard calibration curve (Fig. 5a). With
295
increasing imidacloprid concentration, the fluorescence intensity of the
296
T-line gradually decreased (Fig. 5b). Quantitative measurement was
297
further conducted by the fluorescent strip reader. Results showed that the
298
IC50 of LFICS for the detection of imidacloprid was 0.104 ng/mL, and the
299
linear range was 0.012–0.88 ng/mL (IC20–IC80). For semi-quantitative
300
visual detection, we set the cutoff value (visual LOD) at 0.5 ng/mL for
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imidacloprid, at which concentration the signal intensity of the T-line was
302
faint (Fig. 5c).
303
Selectivity of LFICS
304
The selectivity of the test strip was evaluated by testing the cross
305
reactivity (CR) of the assay with seven other neonicotinoid insecticides
306
(dinotefuran,
307
thiamethoxam, and clothianidin). The LFICS had a high degree of
308
cross-reactivity with imidaclothiz (61.2%) and clothianidin (31.5%),
309
whereas it displayed negligible cross-reactions to the other five
310
neonicotinoids (CR < 1.5%) (Table 1). This indicated that the strips could
311
also be used to detect imidaclothiz (IC20 to IC80: 0.028 to 1.13 ng/mL,
312
limit of visual detection: 0.5 ng/mL) and clothianidin (IC20 to IC80: 0.039
313
to 2.90 ng/mL, limit of visual detection: 1 ng/mL) with good sensitivities.
314
For imidaclothiz and clothianidin, the calibration curves and photos under
315
365 nm excited UV light are shown in Fig. S2 and Fig. S3. Using the strip
316
reader
317
neonicotinoids were higher than those from most studies (Table 2).
318
Tea matrix effects on LFICS
for
nitenpyram,
quantification,
acetamiprid,
the
imidaclothiz,
assay sensitivities
to
thiacloprid,
the
three
319
Sample pretreatment prior to analysis by UPLC-MS/MS was tedious.
320
A rapid, simple and on-site sample-pretreatment method with a
321
high-extraction rate was needed. We diluted the tea infusion with assay
322
buffer to reduce the matrix effect. Results shown in Fig. 6 indicate that
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different tea matrices can have different influences on assay performance,
324
which was consistent with the findings of Jiao et al.6. Excess dilution can
325
reduce the assay’s sensitivity. A 160-fold dilution of green tea, 80-fold
326
dilution of black tea, and 20-fold dilution of oolong tea were selected for
327
LFICS to produce a negligible matrix effect.
328
Considering the dilution factors for matrix effects, the visual LOD of
329
imidacloprid and imidaclothiz were 0.01–0.08 mg/kg in teas and that of
330
clothianidin was 0.02–0.16 mg/kg in tea. Thus, the newly-developed
331
LFICS had a desirable naked-eye sensitivity that satisfied the
332
requirements of the maximum residue limits (MRLs) for imidacloprid
333
(0.5 mg/kg) and imidaclothiz (3 mg/kg) by the National Food Safety
334
Standard of China (GB2763-2016) and for clothianidin (0.7 mg/kg) by
335
the European Union (EU). Additionally, with the aid of the strip reader,
336
the LOD of imidacloprid in tea was 8 × 10-6–6.4 × 10-4 mg/kg, which
337
could satisfy the EU MRL requirement for imidacloprid (0.05 mg/kg).
338
339
Analysis of spiked samples by LFICS
340
Data showing the accuracy and precision of spiked samples are
341
presented in Tables 3, S2, and S3. The spiked levels were selected to be
342
between the assay working range and naked-eye sensitivity. Acceptable
343
recovery of 70.71%–110.78% was obtained, with inter-day CVs of
344
6.89%–11.67% and intra-day CVs of 1.94%–12.49%. The LFICS would
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be suitable for the rapid detection of imidacloprid, imidaclothiz, and
346
clothianidin in green tea, black tea and oolong tea, with desirable
347
sensitivities meeting the MRL requirements.
348
Analysis of authentic samples
349
To further evaluate the reliability of the strip test, eight tea samples
350
(S1–S8) were collected from different tea-producing areas, detected by
351
LFICS, and then confirmed by UPLC-MS/MS.
352
Since the broadly-specific antibody was unable to completely
353
distinguish imidaclothiz, imidacloprid and clothianidin from each other, a
354
standard calibration curve of three mixed pesticides was established to
355
determine the total amount of the three pesticides in the samples. The
356
mixture curve was in accordance with the individual curves of
357
imidacloprid, imidaclothiz, and clothianidin (Fig. S5) when the total
358
concentration of neonicotinoids was in the detection level. Therefore, the
359
mixture curve was used to calculate the unknown concentration of
360
neonicotinoids in the tea samples. As shown in Table 4, 3 out of the 8
361
samples were positive, with concentrations of 0.03–0.125 mg/kg. With
362
the help of UPLC-MS/MS confirmation, the exact types of pesticides
363
were identified (Table 4). The results detected by the LFICS were
364
consistent with those from UPLC-MS/MS.
365
We developed an LFICS employing quantum dots as fluorescent
366
probes for the rapid detection of neonicotinoid insecticides. The major
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367
ratio of quantum dot to antibody, the pH of assay buffer, sensitivity,
368
selectivity, matrix effects, and the assay’s accuracy and reliability were
369
investigated. Using the strip reader, we found that the sensitivities of the
370
QD-based LFICS for the detection of three neonicotinoids (imidacloprid,
371
imidaclothiz, and clothianidin) were higher than those of previously
372
reported
373
neonicotinoids can fully meet their MRLs on teas in China and partly
374
reach the EU MRLs.
immunoassays.
Moreover,
the
visual
LOD
of
three
375
LFICS can simultaneously detect three neonicotinoids, which was
376
convenient for screening pesticides commonly used for insect control on
377
tea plants. The rapid, sensitive and portable QD-based LFICS could be
378
applied to screen neonicotinoid residues by the naked eye on-site or under
379
outside laboratory conditions. This will contribute to the regulation of
380
neonicotinoid use on tea and other agricultural products and reduce the
381
risk of human exposure to neonicotinoid residues.
382
383
Acknowledgements
384
This research was financially supported by National Key R&D
385
Program of China (2017YFF0210200), National Natural Science
386
Foundation of China (31401768) and the Agricultural Project for Public
387
Technology Research in Zhejiang province (2016C32004).
388
389
Conflict of interests
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391
392
The authors have declared no conflict of interests.
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511
512
513
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515
516
517
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Figures
523
524
525
Fig. 1. The display of quantum-dot (QD)-labelled lateral flow
526
immunochromatographic strip (LFICS) under UV excitation and placed
527
in a fluorescent reader.
528
16
14
IC50 (ng/mL)
12
T/C in Control group
18
10
4
2
0
1:5
1:10
1:15
Molar Ratio
8
6
4
2
0
1:5
1:10
1:15
Molar Ratio
529
530
Fig. 2. Effects of the molar ratio of quantum dots to imidacloprid
531
antibody (1:5, 1:10, 1:15) on the assay performance. Bar, ± SD (n=4)
532
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5
T/C
IC50
4
4
3
3
2
2
1
1
0
IC50 (ng/mL)
T-line to C-line Ratio
5
Page 26 of 34
0
Millipore HiFlow-135
Satorius CN-140
Millipore HiFlow-180
Working Membrane
533
534
Fig. 3. The sensitivity (IC50) and T/C ratio of strips based on different
535
working membranes. Bar, ± SD (n=4)
536
1.5
1.5
1.0
1.0
0.5
0.5
0.0
IC50 ( ng / mL )
T-line to C-line Ratio
T/C
IC50
0.0
pH 7.0
pH 7.5
pH 8.0
pH 8.5
Levels of pH in Working buffer
537
538
Fig. 4. Effects of pH levels (pH 7.0, 7.5, 8.0, 8.5) in working buffer on
539
the assay performance. Bar, ± SD (n=4)
540
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T/C
T-line to C-line Ratio
1.0
0.5
0.0
1E-3
0.01
0.1
1
10
100
Concentration of Imidacloprid (ng/mL)
541
(a)
542
543
(b)
544
(c)
545
Fig. 5. (a) The calibration curve of imidacloprid in borate buffer (10 mM,
546
pH 7.5). Bar, ± SD (n=4) (b) The photo of test strips with different
547
concentrations (0.003 - 100 ng/mL) of imidacloprid under 365 nm UV
548
excitation. (c) Test strips treated with borate buffer (imidacloprid-free)
549
and borate buffer containing imidacloprid (0.5 ng/mL) under 365 nm UV
550
excitation.
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Green Tea
1.0
0.5
1.0
0.5
1E-3
0.01
0.1
1
10
Concentration of Imidacloprid (ng/mL)
100
BB Buffer
10 - fold
20 - fold
40 - fold
80 - fold
160 - fold
1.0
0.5
0.0
0.0
0.0
1.5
T-line to C-line Ratio
1.5
Oolong Tea
BB Buffer
10 - fold
20 - fold
40 - fold
80 - fold
160 - fold
1.5
T-line to C-line Ratio
T-line to C-line Ratio
Black Tea
BB Buffer
10 - fold
20 - fold
40 - fold
80 - fold
160 - fold
2.0
Page 28 of 34
1E-3
0.01
0.1
1
10
Concentration of Imidacloprid (ng/mL)
100
1E-3
0.01
Fig. 6. Matrix effects of different tea types on the assay performance. Bar, ±SD (n=4)
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0.1
1
10
Concentration of Imidacloprid (ng/mL)
100
Page 29 of 34
Journal of Agricultural and Food Chemistry
Tables
Table 1. The cross reactivity (CR) of the strip test to eight neonicotinoids.
Analytes
IC50 (ng/mL)
CR(%)
0.104
100
Imidaclothiz
0.17
61.2
Clothianidin
0.33
31.5
Thiacloprid
7.885
1.31
Nitenpyram
8.701
1.2
Acetamiprid
14.101
0.7
Dinotefuran
>1000
<0.01
Thiamethoxam
>1000
<0.01
Imidacloprid
Structure
Cl
N
CH2
N
N NO
2
NH
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Table 2. Comparison of the sensitivity of QD-based strip tests for the detection of
imidacloprid, imidaclothiz, clothianidin with other reported immunoassays.
Analysis method
Analyte
Sensitivity in assay Application
buffer (ng/mL)
sample
Reference
ELISA
Clothianidin
25.6 (IC50)
10
Colloidal gold
Clothianidin
8 (visual LOD)
ELISA
Imidaclothiz
58.16 (IC50)
River water, soil,
cabbage, tomato,
greengrocery
Tomato, pear,
cabbage
Chemiluminescenc
e immunoassay
ELISA
Imidaclothiz
1.8 (IC10)
29
Clothianidin
4.4 (IC50)
ELISA
Imidacloprid
Clothianidin
2.7 (IC50)
>5000 (IC50)
Colloidal
gold-based strips
Imidacloprid
0.5 (visual LOD)
Signal
amplified
colloidal
gold-based strips
Quantum dot-based
ELISA
Imidacloprid
10 (visual LOD)
Tomato, cabbage,
rice
Tomato, cucumber,
apple
Pond water, rice
field water, canal
water, fish pond,
Dushu lake water
Cucumber, tomato,
lettuce, apple,
orange
Chinese cabbage
Clothianidin
12.5 (IC50)
22
Quantum
dot-based strip
Imidacloprid
0.104, 0.5 (IC50,
visual LOD)
0.17, 0.5 (IC50, visual
LOD)
0.33, 1 (IC50, visual
LOD)
Water, soil,
cabbage, rice,
tomato
Green tea,
black tea,
oolong tea
Imidaclothiz
Clothianidin
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30
31
13
14
This article
Page 31 of 34
Journal of Agricultural and Food Chemistry
Table 3. The accuracy and precision of the developed LFICS for the detection of
imidacloprid in teas a.
Tea types
Spiked
level
(ng/kg)
Green Tea
Black Tea
Oolong Tea
a
3.2
80
128
1.6
40
64
0.4
10
16
Intra-batch
(n=4)
Inter-batch
(n=4)
Mean ± SD
(ng/g)
CV
(%)
Recovery
(%)
2.68±0.29
72.94±7.64
119.99±12.21
1.42±0.12
39.62±4.62
70.90±4.89
0.30±0.03
9.17±0.93
14.54±1.42
10.67
10.47
10.18
8.73
11.67
6.89
9.27
10.12
9.76
83.87
91.17
93.74
88.91
99.05
110.78
75.43
91.67
90.89
Mean ± SD
(ng/g)
2.64±0.33
80.58±4.74
122.32±7.73
1.35±0.10
28.28±2.94
61.08±4.31
0.42±0.03
9.57±0.76
16.34±1.42
CV
(%)
12.49
5.88
6.32
7.57
10.38
7.05
6.5
7.94
8.66
Data are mean ± SD from quadruplicate samples at each spiked concentration of
imidacloprid.
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Recovery
(%)
82.63
100.72
95.56
84.58
70.71
95.44
104.44
95.66
102.14
Journal of Agricultural and Food Chemistry
Table 4. Eight tea samples analyzed by LFICS and UPLC-MS/MS (n=4) a
Samples
S1
S2
S3
S4
S5
S6
S7
S8
Determined by
LFICS
(mg/kg)
0.030±0.002
0.080±0.01
ND b
0.125±0.01
ND
ND
ND
ND
Determined by UPLC-MS/MS
(mg/kg)
0.032±0.004 (imidacloprid)
0.082±0.01 (imidacloprid)
ND
0.122±0.01 (imidacloprid)
ND
ND
ND
ND
a
All data are presented as mean±SD from quadruplicate well analysis of each sample.
b
ND means not determined.
Scheme 1. The direct competitive immunoassay of lateral flow
immunochromatographic strip for the detection of neocotinoids in teas.
Coating antigen was imidacloprid-OVA, analyte was imidacloprid,
imidaclothiz or clothianidin, antibody was imidacloprid monoclonal
antibody.
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