Rapid screening of flonicamid residues in environmental and agricultural samples by a sensitive enzyme immunoassay

Article abstract of DOI:10.1016/j.scitotenv.2016.02.017

A fast and sensitive polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed for the analysis of flonicamid in environmental and agricultural samples. Two haptens of flonicamid differing in spacer arm length were synthesized and conjugated to proteins to be used as immunogens for the production of polyclonal antibodies. To obtain most sensitive combination of antibody/coating antigen, two antibodies were separately screened by homologous and heterologous assays. After optimization, the flonicamid ELISA showed that the 50% inhibitory concentration (IC₅₀ value) was 3.86 mg L^-1, and the limit of detection (IC₂₀ value) was 0.032 mg L^-1. There was no cross-reactivity to similar tested compounds. The recoveries obtained after the addition of standard flonicamid to the samples, including water, soil, carrot, apple and tomato, ranged from 79.3% to 116.4%. Moreover, the results of the ELISA for the spiked samples were largely consistent with the gas chromatography (R² = 0.9891). The data showed that the proposed ELISA is an alternative tool for rapid, sensitive and accurate monitoring of flonicamid in environmental and agricultural samples.

Full text of DOI:10.1016/j.scitotenv.2016.02.017

Science of the Total Environment 551–552 (2016) 484–488
Contents lists available at ScienceDirect
Science of the Total Environment
journal homepage: www.elsevier.com/locate/scitotenv
Rapid screening of flonicamid residues in environmental and agricultural
samples by a sensitive enzyme immunoassay
a
a
a
b
a
a
Zhenjiang Liu a, , Zhen Zhang , Gangbing Zhu , Jianfan Sun , Bin Zou , Ming Li , Jiagao Wang
⁎
a
School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, China
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China
b
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
• The antibody of flonicamid was
obtained.
• A high-throughput, selective and simple
ELISA for flonicamid was developed.
• The results of ELISA for the spiked sam-
ples were largely consistent with the
gas chromatography method.
• This methodology appeared to be
useful as a screening method prior to
flonicamid analysis.
a r t i c l e i n f o
a b s t r a c t
Article history:
A fast and sensitive polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed for
the analysis of flonicamid in environmental and agricultural samples. Two haptens of flonicamid differing in
spacer arm length were synthesized and conjugated to proteins to be used as immunogens for the production
of polyclonal antibodies. To obtain most sensitive combination of antibody/coating antigen, two antibodies
were separately screened by homologous and heterologous assays. After optimization, the flonicamid ELISA
showed that the 50% inhibitory concentration (IC50 value) was 3.86 mg L−1, and the limit of detection (IC20
value) was 0.032 mg L−1. There was no cross-reactivity to similar tested compounds. The recoveries obtained
after the addition of standard flonicamid to the samples, including water, soil, carrot, apple and tomato, ranged
from 79.3% to 116.4%. Moreover, the results of the ELISA for the spiked samples were largely consistent with
the gas chromatography (R2 = 0.9891). The data showed that the proposed ELISA is an alternative tool for
rapid, sensitive and accurate monitoring of flonicamid in environmental and agricultural samples.
© 2016 Elsevier B.V. All rights reserved.
Received 15 December 2015
Received in revised form 2 February 2016
Accepted 2 February 2016
Available online xxxx
Editor: D. Barcelo
Keywords:
Flonicamid
Hapten
Polyclonal antibody
Enzyme-linked immunosorbent assay
Environmental and agricultural
1. Introduction
Flonicamid (N-cyanomethyl-4-trifluoromethylnicotinamide) is a
novel selective systemic pesticide (Tomlin, 2003), which has been
widely applied to rice, leafy vegetables, tomato and tea to control
⁎
Corresponding author.
E-mail address: lzj1984@ujs.edu.cn (Z. Liu).
http://dx.doi.org/10.1016/j.scitotenv.2016.02.017
0048-9697/© 2016 Elsevier B.V. All rights reserved.

Z. Liu et al. / Science of the Total Environment 551–552 (2016) 484–488
485
noxious insects, with high effectiveness against aphids and other suck-
2.3. Buffers and solutions
ing insects (Morita et al., 2000; Hengel and Miller, 2007). As a result,
flonicamid is present in river water, soil and agricultural products. To
protect consumers from risks related to flonicamid residue, maximum
residue limits (MRLs) of flonicamid in agricultural samples have been
established by the USA (0.45 mg kg−1 for carrot) and Japan
(0.4 mg kg−1 for tomato and 1 mg kg−1 for apple) (Xie et al., 2011).
There are no suggested MRLs for flonicamid in China.
The following buffers were used: (A) coating buffer, 0.05 mol L−1
carbonate-buffered saline (CBS), pH 9.6; (B) blocking buffer, 5 g skim
milk in 100 mL of phosphate-buffered saline (PBS, 0.01 mol L−1
,
pH 7.4); (C) washing buffer, PBS containing 0.05% Tween-20 (PBST);
(D) the TMB solution contained 0.4 mmol L−1 TMB and 3 mmol L−1
H2O2 in citrate buffer (pH 5.0).
Several instrument-based detection methods for flonicamid have
been developed, such as gas chromatography (GC) (Xie et al., 2011;
Shi et al., 2015), high-performance liquid chromatography (HPLC)
(Ma et al., 2015) and high-performance liquid chromatography–mass
spectrometry (HPLC–MS) (Chen, 2002; Zywitz et al., 2003; Hengel and
Miller, 2007; Chen et al., 2012; Ko et al., 2014). Although these methods
are characterized by low limits of detection and high precision and sen-
sitivity, they cannot meet the needs of the high-throughput, rapid,
screening of large numbers of environmental and agricultural samples.
Enzyme-linked immunosorbent assay (ELISA) fulfills these require-
ments and has become a reliable analytical tool for rapid screening anal-
ysis (Liu et al., 2013). ELISA has been successfully used to detect
contaminates, including toxins (Sheng et al., 2012; Kawatsu et al.,
2014), antibiotics (Adrian et al., 2012; Peng et al., 2012; Sheng et al.,
2013; Wang et al., 2015) and pesticides (Gurbuz et al., 2009; Liu et al.,
2011; Watanabe et al., 2011; Liu et al., 2013; Navarro et al., 2013;
Abad-Fuentes et al., 2014; Hua et al., 2015). However, ELISA of
flonicamid has not been reported. In this paper, a rapid and sensitive
ELISA was developed for the detection of flonicamid residues in envi-
ronmental and agricultural samples based on polyclonal antibodies.
Furthermore, the ELISA performance was evaluated with conventional
GC-NPD in terms of precision and accuracy using spiked samples.
2.4. Hapten synthesis
The structure and synthesis route of 4-(trifluoromethyl)nicotinoyl
chloride (A), Hapten 1 and Hapten 2 are shown in Fig. 1.
Compound A: 4-(trifluoromethyl)nicotinic acid [2 g (9.7 mmol)] and
toluene (30 mL) were added to a 100-mL round-bottomed flask and
were cooled in an ice bath. Then, 5.3 g (44.6 mmol) of SOCl2 was slowly
added to the solution. After the addition, the temperature was increased
to 45 °C for 2 h and was then gradually increased to 80 °C for 1 h. Finally,
unreacted reagents were removed by a rotary evaporator, and brown
liquid A (1.87 g) was obtained.
Hapten 1: A mixture of 1.3 g (175 mmol) of aminoacetic acid and
17.65 g (175 mmol) of triethylamine in 200 mL of toluene was stirred
and cooled in an ice bath. Then, liquid A was added to the solution slow-
ly, and the reaction mixture was stirred at room temperature for 2 h.
The reaction mixture was moved into a separating funnel, 30 mL
water was added, and the organic phase was separated, dried over an-
hydrous sodium sulfate and evaporated under reduced pressure. This
residue was subjected to column chromatography [silica gel, ethyl ace-
tate:petroleum ether (8:1, v/v)]. Yield: 38%. The product was character-
ized by ESI-MS and NMR: ESI-MS, m/z: 339 [M + Na]+ 1H NMR
.
(400 MHz, DMSO-d6), δ: 4.3 (s, 2H, CH2), 7.58–7.60 (d, 1H, CH), 8.66–
8.68 (m, H, CH), 8.77 (s, H, CH).
2. Materials and methods
Hapten 2: This hapten was synthesized using A and 4-aminobutyric
acid. Yield: 48%. The product was characterized by ESI-MS and NMR:
2.1. Reagents
ESI-MS, m/z: 367 [M + Na]+ 1H NMR (400 MHz, DMSO-d6), δ: 1.73–
.
1.78 (m, 2H, CH2), 2.29–2.51 (m, 2H, CH2), 3.26–3.37 (m, 2H, CH2),
7.81–7.83 (d, 1H, NH), 8.74–8.91 (m, 3H, CH × 3), 12.1 (s, 1H, COOH).
Pesticide-grade flonicamid with a purity of 98.5% was obtained
from Dr. Ehrenstorfer GmbH (Augsburg, Germany). 4-
Trifluoromethylnicotinic acid (TFNA), 4-trifluoromethylnicotinamide
(TFNA-AM) and N-(4-trifluoromethylnicotinoyl) glycine (TFNG) were
purchased from Hayashi Pure Chemical Ind., Ltd (Osaka, Japan). Bovine
serum albumin (BSA), ovalbumin (OVA), Freund's complete and incom-
plete adjuvants, goat anti-rabbit IgG-horseradish peroxidase (GAR-
HRP), polyoxyethylene sorbitan monolaurate (Tween-20), 3,3′,5,5′-
tetramethylbenzidine.
(TMB), N-hydroxysuccinimide (NHS), N,N′-dicyclohexylcarbodiimide
(DCC) and isobutyl chloroformate were purchased from Sigma Chemi-
cal Co. (Shanghai, China). Aminoacetic acid and 4-aminobutyric acid
were purchased from Aldrich (Milwaukee, USA). Acetone, acetonitrile,
toluene, ethyl acetate, petroleum ether, SOCl2, NaHCO3, NaCl, Na2CO3
and so on were purchased from Beijing Chemical Reagent Co., Ltd (Bei-
jing, China). All reagents and solvents were analytical grade.
2.5. Synthesis and identification of hapten–protein conjugates
Two flonicamid haptens were coupled with BSA using the active
ester method to produce an immunogen and were conjugated with
OVA by the mixed anhydride method to produce a coating antigen (Li
et al., 2014). The conjugates were dialyzed against PBS for 72 h at 4 °C
and were stored at −20 °C. The conjugates were confirmed by UV–Vis
spectroscopy. The number of hapten molecules per molecule of protein
(hapten density) of the conjugate was estimated directly by the molar
absorbance at 280 nm (Liu et al., 2011).
À
Á
Hapten density ¼
ε
conjugate−εprotein =εhapten
2.6. Immunization and antibody preparation
2.2. Instruments
According to the method described by Shan et al. (1999), four male
New Zealand white rabbits (approximately 2 kg per rabbit) were divid-
ed into two groups and were immunized for Hapten 1-BSA and Hapten
2-BSA via intraperitoneal injection. The rabbits had free access to drink-
ing water and commercial standard laboratory diet (CZZ, Nanjing,
China) and were housed according to the EEC 609/86 Directives regulat-
Nuclear magnetic resonance (NMR) spectra were recorded on a DRX
500 spectrometer (Bruker, Germany). Mass spectral (MS) data were ob-
tained with a LC-MSQDECA (Finnigan, USA). Ultraviolet spectra were re-
corded on a DU 800 spectrophotometer (Beckman, USA). The 96-well
polystyrene microplates (Maxisorp) were purchased from Nunc
(Roskilde, Denmark). Absorbance was read with an Infinite M200 mi-
crotiter plate reader (Tecan, Switzerland) at 450 nm, and the ELISA
plates were washed with a Wellwash Plus (Thermo, USA). The
flonicamid ELISA was confirmed with an Agilent 7890A gas chromato-
graph (Agilent, USA).
ing the welfare of experimental animals. The immunogen (1 mg kg−1
)
dissolved in physiological saline was emulsified with an equal volume
of Freund's complete adjuvant and was injected intradermally at multi-
ple sites on the back of each rabbit. After the first injection, four boosting
injections were given at 2 week intervals with the immunogen
(1.5 mg kg−1) in Freund's incomplete adjuvant (1:1 v/v). The rabbits

486
Z. Liu et al. / Science of the Total Environment 551–552 (2016) 484–488
Fig. 1. Synthesis route of flonicamid haptens.
were bled from the ear vein using a 1-mL syringe after each boosting in-
jection. Eight days after the last injection, blood samples were obtained
from each rabbit's heart. The antiserum was centrifuged and purified by
salting out with caprylic acid–ammonium sulfate (Li et al., 2008) and
was stored at −20 °C.
and local supermarket of Zhenjiang (China). Each analysis was per-
formed in triplicate.
For water samples, simple filtration with filter paper was performed.
The filtered water samples were spiked with flonicamid standard solu-
tion at 0.05, 0.5 and 5 mg L−1, and were then allowed to stand in the
dark at room temperature for 24 h. The spiked water samples were di-
rectly analyzed by ELISA.
2.7. ELISA protocol
For soil and agricultural samples, samples (10 g) were spiked with
flonicamid at various levels (0.1–10 mg kg−1) and were then allowed
to stand in the dark at room temperature for 24 h. The samples were
then shaken with 20 mL acetonitrile and 10 mL water. After ultrasonic
extraction for 10 min, each suspension was centrifuged for 10 min at
4000 rpm. The supernatant was placed in a separating funnel with 5 g
of NaCl and was vigorously shaken. The organic phase (2 mL) was evap-
orated under vacuum. The residue was dissolved in 4 mL of PBS contain-
ing 10% methanol and was analyzed by ELISA. The recoveries and
relative standard deviation (RSD) were calculated. The spiked samples
were prepared and performed by the same operators.
Microplates were coated with coating antigen (100 μL per well, in
CBS) overnight at 4 °C. The plates were washed three times with PBST.
The binding sites not occupied by coating antigen were blocked with
200 μL of 5% skim milk per well for 1 h at 37 °C. After another washing
step with PBST, flonicamid standards or sample extract (50 μL per
well) and antibody diluted in assay buffer (50 μL per well) were
added to the coated wells and incubated for 1 h. Following a further
wash, 100 μL of diluted GAR-HRP was added and incubated for 1 h at
37 °C. After the plates were washed again, 100 μL per well of TMB solu-
tion was added and incubated for 15 min. Finally, 2 mol L−1 of sulfuric
acid (50 μL per well) was added, and the absorbance was measured at
450 nm. The measurement was performed three times in triplicate
wells.
2.10. Evaluation of the assay by GC
The standard curve for flonicamid was obtained using (B/B0) % ver-
sus the concentration of flonicamid and was fitted to a four-parameter
logistic equation.
To test the effectiveness of the developed ELISA, the spiked samples
were simultaneously analyzed using ELISA and a GC- nitrogen-
phosphorus detector (NPD). For GC-NPD, the samples were extracted
as described above (ELISA procedure). The extracted residue was puri-
fied with a Florisil solid-phase extraction column. Then, the purified ex-
tracts concentrated under vacuum were dissolved with acetone and
were analyzed by GC-NPD (Shi et al., 2015).
The GC-NPD analysis was performed on a HP-5-fused silica capillary
column (30 m × 320 μm × 0.25 μm). The GC conditions were 120 °C for
1 min, a temperature increase to 280 °C at a rate of 25 °C min−1 and held
for 5 min; a carrier gas (N2) flow rate of 3.0 mL min−1; an injection tem-
perature of 280 °C using the splitless mode; and a detector temperature
of 340 °C (Shi et al., 2015).
% (B/B0) was calculated by the following equation:
%ðB=B0Þ ¼ ½ðAx−AminÞ=ðAmax−AminÞꢀ ꢁ 100
where Ax is the absorbance of the sample, Amax is the absorbance in the
absence of the analyte, and Amin is the absorbance of the background.
2.8. Cross-reactivity
The cross-reactivity (CR) was studied using standard solutions of
flonicamid and some analogues. CR was calculated as follows.
CR ð%Þ ¼ ðIC50 of flonicamid=IC50 of analoguesÞ ꢁ 100:
Table 1
Homologous and heterologous ELISA for flonicamid.
2.9. Recovery
Antibody
Coating antigen
Amax
IC50 (mg L−1
)
Amax/IC50
To evaluate the accuracy and precision of the ELISA, the recoveries of
spiked samples (pond water, rice field water, canal water, rice field soil,
vegetable field soil, carrot, apple and tomato) were studied. Eight envi-
ronmental and agricultural samples were collected from the suburbs
Hapten 1-Ab
Hapten 1-OVA
Hapten 2-OVA
Hapten 1-OVA
Hapten 2-OVA
1.15
1.26
1.06
1.01
5.63
7.39
7.32
3.87
0.20
0.17
0.14
0.26
Hapten 2-Ab

Z. Liu et al. / Science of the Total Environment 551–552 (2016) 484–488
487
Table 3
Recovery of flonicamid in spiked samplesa (n = 3).
Sample
Spiked concentration
Dilution
times
Mean recovery
SD (%)
RSD
(%)
(mg L−1, mg kg−1
)
Rice paddy water
5
0.5
0.05
5
0.5
0.05
5
0.5
0.05
10
1
0.1
10
1
0.1
10
1
0.1
10
1
0.1
10
1
0
0
0
2
2
2
2
2
88.3 4.7
95.8 6.9
79.7 6.4
89.1 5.3
81.3 7.5
98.1 6.3
90.6 5.1
85.4 7.8
80.4 6.7
112 3.9
99.8 3.6
111.3 5.4
98.7 8.5
97.5 6.1
99.8 7.2
116.4 7.9
100.6 5.9
98.3 6.2
95.3 8.5
88.7 6.9
89.3 5.6
86.3 4.7
79.3 8.4
93.5 5.3
5.3
7.2
8.0
5.9
9.2
6.4
5.6
9.1
8.3
3.5
3.6
4.9
8.6
6.3
7.2
6.8
5.9
6.3
8.9
7.8
6.3
5.4
10.6
5.7
Pond water
Canal water
Rice paddy soil
Vegetable field soil
Carrot
Apple
Tomato
Fig. 2. Standard curve of the optimized ELISA for flonicamid (the combination of Hapten 2-
Ab and Hapten 2-OVA was used).
0.1
a
100 mg L−1 and 50 mg L−1 of flonicamid standard solution were used for spiking.
3. Results and discussion
shown in Table 1. The combination of Hapten 2-Ab and Hapten 2-OVA
produced the lowest IC50 value, and the highest Amax/IC50 ratio was
used for the ELISA.
3.1. Identification of artificial antigens and coupling ratio
The UV–Vis spectra showed qualitative difference between the hap-
ten, carrier proteins and conjugates, especially at 280 nm, and also
showed quantitative difference that the coupling ratios of hapten to
protein were 31:1, 35:1, 7:1 and 8:1 for Hapten 1-BSA, Hapten 2-BSA,
Hapten 1-OVA, and Hapten 2-OVA, respectively. The results indicated
that the conjugates were coupled successfully.
The application of ELISA to agricultural and environmental samples
requires consideration of several experimental factors, such as the salt
concentration, pH and solvents affecting the performance of the immu-
noassay. In the study, the results indicated that when the PBS buffer
contained 10% methanol and 0.3 mol L−1 Na+ at pH 7.5 (Figs. A.1 to
A.2, see Supplementary Material), the assay exhibited the highest
sensitivity.
3.2. Development of the ELISA
When the combination of Hapten 2-Ab and Hapten 2-OVA, 10%
methanol, 0.3 mol L−1 Na+ and pH 7.5 were used, a competitive stan-
dard curve for flonicamid was obtained by a four-parameter logistic
equation. The results are presented in Fig. 2. The limit of detection
(LOD, IC20 value) and the sensitivity (IC50 value) of the ELISA were
0.032 mg L−1 and 3.86 mg L−1, respectively. The linear working range
determined as the concentrations causing 20–80% inhibition, were
0.032–685.4 mg L−1 for the ELISA.
To develop a sensitive ELISA for flonicamid pesticides, each of the an-
tibodies for analyte recognition was screened by competitive indirect
ELISA using each of the two coating antigens. The IC50 value and Amax
IC50 ratio were used as the primary criteria to evaluate the ELISA perfor-
mance; the lowest IC50 value and the highest ratio of Amax/IC50 indicated
the highest sensitivity (Mercader and Montoya, 1999). The results are
/
Table 2
Cross-reactivities of related compounds in the flonicamid immunoassaya.
Compound
Flonicamid
Structure
IC50 (mg L−1
3.87
)
CR (%)
100
Hapten 2
TFNA-AM
1.26
155
N100
0.03
TFNA
N1000
N1000
b0.01
b0.01
TFNG
a
The combination of Hapten 2-Ab and Hapten 2-OVA was used for the cross-reactivity.

488
Z. Liu et al. / Science of the Total Environment 551–552 (2016) 484–488
Chen, A.W., 2002. Analytical methodology for IKI-220 (F1785) and its major metabolites
3.3. Specificity
in/on peach, potato tuber, and wheat straw. Analytical Report (P-3561M) for FMC
Corporation, Princeton, NJ.
The values of the CR of the antibody with flonicamid and some ana-
logues are shown in Table 2. The ELISA showed negligible CR with its
tested metabolites, and the highest CR was TFNA-AM (0.03%). There-
fore, the results indicated the antibody had high specificity for
flonicamid.
Chen, G., Sun, Y.M., Yang, T., Wu, Y.L., 2012. Simultaneous determination of flonicamid
and its metabolites in cucumbers and apples by liquid chromatography–tandem
mass spectrometry. Chin. J. Chromatogr. 30, 555–559.
Gurbuz, F., Metcalf, J.S., Karahan, A.G., Codd, G.A., 2009. Analysis of dissolved microcystins
in surface water samples from Kovada Lake, Turkey. Sci. Total Environ. 407,
4038–4046.
Hengel, M.J., Miller, M., 2007. Analysis of flonicamid and its metabolites in dried hops by
liquid chromatography–tandem mass spectrometry. J. Agric. Food Chem. 55,
8033–8039.
3.4. Analysis of spiked samples
Hua, X.D., Liu, X.F., Yin, W., Xia, Y.Z., Zhou, Q.J., Lu, Y.W., et al., 2015. A sensitive monoclo-
nal antibody-based enzyme-linked immunosorbent assay for the detection of
bifenthrin in a chemical soil barrier. Sci. Total Environ. 502, 246–251.
Kawatsu, K., Kanki, M., Harada, T., Kumeda, Y., 2014. A highly rapid and simple competi-
tive enzyme-linked immunosorbent assay for monitoring paralytic shellfish poison-
ing toxins in shellfish. Food Chem. 162, 94–98.
Ko, A.Y., El-Aty, A.M.A., Rahman, M.M., Jang, J., Kim, S.W., Choi, J.H., Shim, J.H., 2014. A
modified QuEChERS method for simultaneous determination of flonicamid and its
metabolites in paprika using tandem mass spectrometry. Food Chem. 157, 413–420.
Li, B., Shi, H.Y., Wang, M.H., 2008. An enzyme-linked immunosorbent assay for the detec-
tion of bifenthrin. Chin. J. Anal. Chem. 36, 34–38.
As illustrated in Table 3, the recoveries of flonicamid ranged from
79.3 to 116.4% for the spiked samples (the number of samples was 3),
and the RSDs were less than or equal to 10.6%. Therefore, the accuracy
and precision for the ELISA were satisfactory for the quantitative detec-
tion of flonicamid in agricultural and environmental samples.
3.5. Validation of the assay by GC
Li, M., Hua, X.D., Ma, M., Liu, J.S., Zhou, L.L., Wang, M.H., 2014. Detecting clothianidin res-
idues in environmental and agricultural samples using rapid, sensitive enzyme-
linked immunosorbent assay and gold immunochromatographic assay. Sci. Total En-
viron. 499, 1–6.
Liu, Z.J., Yu, P.M., Fang, S., Fan, J.Q., Wang, M.H., 2011. Development of an enzyme-linked
immunosorbent assay for determination of pretilachlor in water and soil. Ecotoxicol.
Environ. Saf. 74, 1595–1599.
Liu, Z.J., Yan, X., Xu, X.Y., Wang, M.H., 2013. Development of a chemiluminescence
enzyme-linked immunosorbent assay for the simultaneous detection of imidaclothiz
and thiacloprid in agricultural samples. Analyst 138, 3280–3286.
Ma, M., Chen, M., Wang, M.H., 2015. Quantitative analysis of flonicamid by HPLC. World
Pestic. 37, 51–53.
The spiked samples were analyzed by ELISA and GC. The equation of
the line obtained from the linear regression of the combined ELISA and
GC data (Table A.1, see Supplementary Material) for flonicamid was
y = 0.9609x − 0.1055 (R2 = 0.9891, n = 24). The result of the correla-
tion further demonstrated the reliability of the proposed ELISA method.
4. Conclusions
In summary, a specific and accurate ELISA based on a polyclonal an-
tibody was successfully developed to detect flonicamid in agricultural
and environmental samples. The antibody showed high sensitivity and
specificity, with an LOD value of 0.032 mg L−1. The cross-reactivity for
some analogs was b0.03%. Analysis of spiked samples indicated that
the specificity and accuracy of the ELISA were ideal and in good agree-
ment with the GC measurements. Therefore, the proposed ELISA is a fea-
sible quantitative/screening method for flonicamid in agricultural and
environmental samples due to its sensitivity and simplicity, rapidity,
lower expenses and high sample throughput.
Mercader, J.V., Montoya, A., 1999. Development of monoclonal ELISAs for azinphos-
methyl. 2. Assay optimization and water sample analysis. J. Agric. Food Chem. 47,
1285–1293.
Morita, M., Ueda, T., Yoneda, T., Koyanagi, T., Murai, S., Matsuo, N., et al., 2000. Ishihara
Sangyo Kaisha, Ltd. IKI-220—a novel systemic aphicide. BCPC Conference-Pests & Dis-
ease, p. 5965.
Navarro, P., Pérez, A.J., Gabaldón, J.A., Núñez-Delicado, E., Puchades, R., Maquieira, A., et al.,
2013. Detection of chemical residues in tangerine juices by a duplex immunoassay.
Talanta 116, 33–38.
Peng, D.P., Ye, S.Q., Wang, Y.L., Chen, D.M., Tao, Y.F., Huang, L.L., et al., 2012. Development
and validation of an indirect competitive enzyme-linked immunosorbent assay for
the screening of tylosin and tilmicosin in muscle, liver, milk, honey and eggs.
J. Agric. Food Chem. 60, 44–51.
Shan, G.M., Stoutamire, D.W., Wengatz, I., Gee, L.H., Hammock, B.D., 1999. Development of
an immunoassay for the pyrethroid insecticide esfenvalerate. J. Agric. Food Chem. 47,
2145–2155.
Acknowledgments
Sheng, Y.J., Jiang, W.X., Saeger, S.D., Shen, J.Z., Zhang, S.X., Wang, Z.H., 2012. Development
of a sensitive enzyme-linked immunosorbent assay for the detection of fumonisin B1
in maize. Toxicon 60, 1245–1250.
Sheng, W., Yang, L., Wang, J.P., Zhang, Y., Wang, S., 2013. Development of an enzyme-
linked immunosorbent assay for the detection of gentamycin residues in animal-
derived foods. LWT Food Sci. Technol. 50, 204–209.
Shi, K.W., Tang, C.F., Li, W., Li, L., 2015. Residue and dietary risk assessment of flonicamid
in apple. Agrochemicals 54, 674–677.
Tomlin, C.D.S., 2003. The pesticide manual. 13th ed. British Crop Protection Council, Sur-
This work was supported by the Natural Science Foundation of Jiang-
su Province (BK20140543), the China Postdoctoral Science Foundation
(2014M561596), the Senior Talent Scientific Research Initial Funding
Project of Jiangsu University (14JDG051), the Project Funded by the Pri-
ority Academic Program Development of Jiangsu Higher Education In-
stitutions (PAPD) and the Jiangsu Collaborative Innovation Center of
Technology and Material of Water Treatment.
rey, UK.
Wang, Z.H., Mi, T.J., Beier, R.C., Zhang, H.Y., Sheng, Y.J., Shi, W.M., et al., 2015. Hapten syn-
thesis, monoclonal antibody production and development of a competitive indirect
enzyme-linked immunosorbent assay for erythromycin in milk. Food Chem. 171,
98–107.
Watanabe, E., Baba, K., Miyake, S., 2011. Analytical evaluation of enzyme-linked immuno-
sorbent assay for neonicotinoid dinotefuran for potential application to quick and
simple screening method in rice samples. Talanta 84, 1107–1111.
Xie, J.J., Chen, J., Wang, L., Jiao, H., Ye, H.Y., Lai, Z.F., 2011. Study on the determination of
flonicamid residues in plant-based foods by gas chromatography. J. Northwest A F
Univ. (Nat. Sci. Ed.) 39, 82–86.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.scitotenv.2016.02.017.
References
Abad-Fuentes, A., Agullo, C., Esteve-Turrillas, F.A., Abad-Somovilla, A., Mercader, J.V., 2014.
Immunoreagents and competitive assays to fludioxonil. J. Agric. Food Chem. 62,
2742–2744.
Adrian, J., Fernández, F., Sánchez-Baeza, F., Marco, M.P., 2012. Preparation of antibodies
and development of an enzyme-linked immunosorbent assay (ELISA) for the deter-
mination of doxycycline antibiotic in milk samples. J. Agric. Food Chem. 60,
3837–3846.
Zywitz, D., Anastassiades, M., Scherbaum, E., 2003. Simultaneous determination of
neonicotinoid insecticides in fruits and vegetables by LC–MS and LC–MS–MS: meth-
odology and residue data. Dtsch. Lebensm.-Rundsch. 99, 188–196.