Lateral flow immunoassay for the simultaneous detection of fipronil and its metabolites in food samples

Article abstract of DOI:10.1016/j.foodchem.2021.129710

We developed a sensitive and rapid lateral flow immunochromatographic (LFI) assay for the simultaneous detection of fipronil and its metabolites in eggs and cucumbers using gold nanoparticle (GNP)-labeled monoclonal antibodies (mAbs). Anti-fipronil mAbs (

Full text of DOI:10.1016/j.foodchem.2021.129710

Food Chemistry 356 (2021) 129710
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Lateral flow immunoassay for the simultaneous detection of fipronil and its
metabolites in food samples
,
,
,
,
,b,*
Jingjing Yaoa b, Zhongxing Wanga b, Lingling Guoa b, Xinxin Xua b, Liqiang Liua
,
,
b
,
,b
Hua Kuanga *, Chuanlai Xua
a State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, People’s Republic of China
b International Joint Research Laboratory for Biointerface and Biodetection, School of Food Science and Technology, Jiangnan University, Wuxi, People’s Republic of
China
A R T I C L E I N F O
A B S T R A C T
Keywords:
We developed a sensitive and rapid lateral flow immunochromatographic (LFI) assay for the simultaneous
detection of fipronil and its metabolites in eggs and cucumbers using gold nanoparticle (GNP)-labeled mono-
clonal antibodies (mAbs). Anti-fipronil mAbs (1B6) were produced using two haptens and identified by heter-
ologous indirect competitive enzyme-linked immunosorbent assay (icELISA) with half maximal inhibitory
concentration (IC50) and limit of detection (LOD) values of 0.46 ± 0.07 and 0.05 ± 0.01 ng mLꢀ 1, respectively.
The developed LFI strip showed high sensitivity and specificity in the detection of fipronil with cut-off and visual
limit of detection (vLOD) values of 10 and 0.25 ng mLꢀ 1, respectively. Furthermore, the application of LFI in the
detection of fipronil-spiked egg and cucumber samples was validated by liquid chromatography tandem mass
spectrometry (LC-MS/MS). Our developed LFI assay is suitable for detection of fipronil and its metabolites in real
samples.
Lateral flow immunoassay
Fipronil and metabolites
Monoclonal antibody
Immunochromatographic test strip
Eggs
Cucumbers
´
1. Introduction
Hagen, Jouanin, Perdu, Martin, & Viguie, 2012). The illegal use of
fipronil in poultry farms resulted in the 2017 fipronil eggs contamina-
Pesticides improve the productivity and profitability of agricultural
products. Fipronil is a highly effective phenylpyrazole insecticide, which
is used in crop pest control, greenhouse (Li et al., 2014), and urban pest
management (Simon-Delso et al., 2015). Fipronil impacts γ-amino-
butyric acid (GABA) receptors and effectively blocks GABA-regulated
chloride channels in the nervous system, paralyzing or killing the
target organism. Exposure to low concentrations of fipronil will induce
cell death (Vidau et al., 2011). Even though fipronil does not accumulate
in abiotic environments, its half-life (T0.5) is 270–555 d in soil (Chawla
et al., 2018). Fipronil can be degraded by photolysis, hydrolysis,
oxidation, and reduction to form four major products: fipronil desul-
finyl, hydroxy-fipronil, fipronil sulfone, and fipronil sulfide (Hainzl,
Cole, & Casida, 1998; Vasylieva et al., 2017) (Supplementary Figure S1).
Studies have reported that bioaccumulation of fipronil and fipronil
sulfone can occur in fatty tissues (Dheeraj, Seema, & Astha, 2014).
Moreover, fipronil is genotoxic (Yildirim & Agar, 2016), and fipronil
desulfinyl, fipronil sulfone, fipronil sulfide are more poisonous than
fipronil (Romero et al., 2016; Roques, Lacroix, Puel, Gayrard, Picard-
tion is an incident in Europe and Asia.
Considering that fipronil and its metabolites in crops and the envi-
ronment can be transferred to foods, Announcement No. 1157 of the
Ministry of Agriculture of P.R. China has made a provision (Zhang et al.,
2016). Therefore, it is imperative to develop a simple, sensitive, and
reliable method for detecting fipronil and its metabolites in foodstuffs.
Fipronil residues have been detected in soil (Tao et al., 2014), water
(Martins, Donato, Prestes, Adaime, & Zanella, 2013), tea (Chen et al.,
2018), honey (Nakajima et al., 2015; Tomasini et al., 2012), eggs (Guo
et al., 2018; Zhang et al., 2016), cake (Guo et al., 2018), and muscle
tissue (Guo et al., 2018; Zhang et al., 2016) by instrumental analysis.
Enzyme-linked immunosorbent assays (ELISA) based on polyclonal an-
tibodies (pAbs), monoclonal antibodies (mAbs), or single-domain anti-
bodies has been developed for the detection of fipronil residues have
been reported. As described by Xu et al. in 2007, the pAbs and mAbs
against fipronil were prepared and used for ELISA with IC50 values of
17.95 and 5.99 ng mLꢀ 1, respectively(Liu, Yan, Dong, Yu, & Xu, 2007).
Then, two ELISAs having IC50 values of 0.58 and 2.6 ng mLꢀ 1 were
* Corresponding authors at: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, People’s Republic of China.
E-mail addresses: murel@163.com (L. Liu), kuangh@jiangnan.edu.cn (H. Kuang).
https://doi.org/10.1016/j.foodchem.2021.129710
Received 11 August 2020; Received in revised form 23 March 2021; Accepted 24 March 2021
Available online 27 March 2021
0308-8146/© 2021 Elsevier Ltd. All rights reserved.

J. Yao et al.
Food Chemistry 356 (2021) 129710
developed with distinct cross-reactivity patterns for structurally related
compounds, such as fipronil sulfide, fipronil detrifluoromethylsulfonyl,
and fipronil desulfinyl, respectively using different haptens and coating
antigens(Vasylieva, Ahn, Barnych, Gee, & Hammock, 2015). Recently,
Hammock and Xu et al. reported that two camel single-domain anti-
bodies (VHHs) F1 and F6 against fipronil and fipronil sulfone were ob-
tained and used to develop ELISAs for the detection of the two
compounds in the sera of black-tailed prairie dogs and rats with LOD
values of 10 and 30 ng mLꢀ 1. The development of more sensitive and
convenient immunoassay for fipronil and its metabolites is a challenge
demanding solution.
2. Materials and methods
2.1. Synthesis of fipronil haptens
As shown in Fig. 1, we searched and designed structure and mech-
anism of fipronil haptens according to Scifinder Scholar (Pojani & Bar-
locco, 2020) with slight modifications (Liu et al., 2007; Vasylieva et al.,
2015; Zhang & Yu, 2016; Zheng & Newman, 2021). Firstly, fipronil
(200 mg) was dissolved in 2.0 mL acetone in a round flask and mixed
with 20 mL of 0.1 mol L-1 aqueous NaOH. The mixture was heated for 24
h at 60 ◦C. After reaching room temperature (RT), the pH of the mixture
was adjusted to 5.0 with 1.0 mol L-1 aqueous HCl. A yellow solid (hapten
FPA) was obtained, filtered under vacuum, dried, and characterized by
HPLC-MS (Fig. 2a) and 1H NMR (Fig. 3a).
In this study, we synthesized two types of fipronil haptens and pre-
pared a highly sensitive and specific mAb against fipronil and its me-
tabolites based on heterologous indirect competitive ELISA (icELISA).
Additionally, we developed a lateral flow immunochromatographic
(LFI) assay with visual limit of detection (vLOD) of 0.25 ng mLꢀ 1 in
determination fipronil. In view of the 2017 fipronil eggs contamination
incident, and cucumbers as common vegetables, the application of the
LFI strip in detection of fipronil in eggs and cucumbers was investigated.
Then, the 5-Amino-1-[2, 6-dichloro-4-(trifluoromethyl)phenyl]-1H-
pyrazole- 3-carbonitrile (compound 1, 64 mg, 0.2 mmol) was dissolved
in acetonitrile (1.0 mL) and mixed with NIS (1.1 mmol) at RT (Fig. 1b).
The mixture was allowed to react for 1 h under reflux. Water was added
and extracted three times with ethyl acetate. The organic phase was
collected, rinsed with saturated NaCl, dried, and concentrated. The in-
termediate 2 was obtained through column chromatography purifica-
tion. Subsequently, reactant 2 (1.0 mmol), methanol (MeOH, 5.0 mL),
Pd(OAc)2 (0.5 mmol), and triethylamine (Et3N, 3.0 mmol) were added
Fig. 1. The mechanism of synthesizing fipronil haptens.
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Food Chemistry 356 (2021) 129710
Fig. 2. The HPLC-MS of fipronil haptens. (a) HPLC-MS of FPA; (b) HPLC-MS of FP.
into 100 mL autoclavable tubes. After the autoclave was closed and N2
was replaced for three times, 0.5 MPa of carbonic oxide (CO) gas was
added, and the mixture was heated for 2 h at 100 ◦C. When the reaction
reached RT, the remaining CO gas was carefully discharged before
opening the reactor. The reaction vessel was cleaned with ethyl acetate,
and the reaction mixture was filtered, concentrated, and purified by
column chromatography (product 3). Finally, product 3 was hydrolyzed
with 0.8 mL of 5% NaOH. The reaction was monitored until completely
hydrolyzed. A solid precipitate was obtained when the pH was adjusted
to 5.0 using 1.0 mol L-1 aqueous HCl, followed by filtration and rinsing.
Finally, the soil hapten FP was obtained and characterized by HPLC-MS
(Fig. 2b) and 1H NMR (Fig. 3b).
assessed by ELISA as previously reported (Chen et al., 2019a, 2019b).
2.4. Optimization of icELISA
The heterologous icELISA was optimized by assessing pH and buff-
ering capacity of the assay solution. Calibration curves were generated
by plotting absorbance against the logarithm of analyte concentration,
from which IC50 values (concentration at which binding of the antibody
to the coating antigen is inhibited by 50%) and LOD (limit of detection,
defined as 10% inhibition) were calculated. In addition, CR of anti-
fipronil mAbs with fipronil desulfinyl, fipronil sulfone, and fipronil
sulfide via icELISA was calculated according to the following formula,
IC50 against fipronil
IC50 against fipronil metabolites and analogues
CR(%) =
× 100%
2.2. Preparation of immunogens and coating antigens
FPA and FP were conjugated with BSA and ovalbumin using EDC.
FPA/FP-BSA and FPA/FP-ovalbumin served as the immunogen and
coating antigen, respectively. In detail, 2.0 mg FPA or 1.6 mg FP was
dissolved in 1.0 mL N, N-dimethylformamide (DMF) and mixed with 4.5
mg EDC with 1.5 mg N-hydroxysuccinimide (NHS) under constant
stirring. The mixture was allowed to react for 0.5 h at 4 ◦C. Subse-
quently, 1 mL of 3.6 g L-1 BSA or ovalbumin in CBS was added into the
2.5. Preparation of GNPs and GNP-mAbs
Gold nanoparticles (GNPs) were synthesized by a modified method
that was previously reported (Chen et al., 2017). First, 100 mL aqueous
HAuCl4⋅4H2O (0.01%, w/v) under boiling was quickly added to 4 mL
freshly prepared aqueous trisodium citrate (1%, w/v). The color of the
mixture changed to wine red. Secondly, after cooling to RT, the solution
was stored at 4 ◦C. The average diameter of GNPs was determined and
characterized by TEM (transmission electron microscope).
◦
mixture dropwise and allowed to react for 3 h at 4 C. Finally, after
dialysis against PBS for 3 d, the product was stored at ꢀ 20 ◦C. The im-
munogens and coating antigens were characterized by ultra-
violet–visible (UV–Vis) spectrophotometry (Supplementary Figure S2).
We prepared GNP-mAbs as previously described (Li, Liu, Song, &
Kuang, 2018; Peng et al., 2016; Suryoprabowo, Liu, Kuang, Cui, & Xu,
2021; Wang et al., 2020). Briefly, the pH of 10 mL GNP solution was
adjusted to 8.0 with 0.1 mol L-1 aqueous K2CO3. Subsequently, mAbs
(0.2 mL) were added dropwise under continuous stirring, and the
mixture was shaken for 1 h RT. The solution was incubated for 2 h under
constant stirring after adding 1 mL of blocking solution (0.5% BSA, w/
v). Subsequently, the mixture was centrifuged for 30 min at 7 × 103 × g
to remove the blocking agent and excess antibodies. The supernatant
was discarded, while the sediment was collected and washed three times
with 0.02 M PBS buffer (0.1% Tween, 0.1% PEG, 5% trehalose, 5%
sucrose, and 0.2% BSA, pH 7.8). Finally, the conjugated probe was
reconstituted with 1 mL resuspension buffer (0.02 M Tris-HCl, 0.1%
Tween-20, 0.1% PEG, 1% PVP, 5% sucrose, 4% trehalose, 2% sorbitol,
2.3. Preparation and identification of anti-fipronil mAbs
Immunization and antiserum detection were performed (Zeng et al.,
2020). The mouse with the highest titer and competitive inhibition was
selected for cell fusion (Wang et al., 2017). Following cell screening by
ELISA and subcloning, the selected monoclonal hybridoma cell line
producing anti-fipronil mAbs was injected into the abdomens of 8-week-
old mice. The mAbs were purified by caprylic acid/ammonium sulphate
precipitation (CA-SA) (Gao et al., 2019; Li, Zhi, Jia, Nie, & Ai, 2018).
Anti-fipronil mAbs were identified by ELISA using a mouse mAb
isotyping kit. The titer, affinity constant, and specificity of mAbs were
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Food Chemistry 356 (2021) 129710
Fig. 3. 1H NMR of fipronil haptens. (a): 1H NMR of FPA; (b) 1H NMR of FP.
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J. Yao et al.
Food Chemistry 356 (2021) 129710
1% mannitol. 0.04% NaN3, and 0.2% BSA) and stored at 4 ◦C.
quantitative curve was generated by plotting logarithm of fipronil con-
centration on the x-axis against T/C values (ratio of the color intensity of
the T line for the positive sample to that for the negative sample, T and C
representing the color intensity of T line and the corresponding C line,
respectively) on the y-axis, from which IC50 value (concentration at
which binding of antibody to the coating antigen was inhibited by 50%),
LOD (limit of detection, defined as 10% inhibition), and detection range
(defined as 10% to 80% inhibition) were calculated.
2.6. Assembly and principle of the LFI strip
The test strip consists of three components: plastic packing, NC
membrane, and pads (sample, probe, and absorbent pads)(Z. Wang,
Guo, Liu, Kuang, & Xu, 2018) (Fig. 4). The NC membrane was sprayed
with FP-ovalbumin on the test line (T line) and GAM-IgG on the control
line (C line). The probe pad was saturated with GNP-mAbs. The sample
pad, probe pad, absorbent pad, and NC membrane were pasted onto the
plastic packing with a 1–2 mm overlap, cut in 3.0 mm wide strips, and
dried for 4 h at 37 ◦C. The strips were sealed and stored at 4 ◦C before
use. The working condition of the LFI strip was optimized, including the
concentration of mAb and coating antigen, GNP-mAb probe resus-
pension (Supplementary Figure S8).
The specificity of the test strip was evaluated. The concentrations of
fipronil desulfinyl (0, 1.0, 2.5, 5.0, 10, 25, and 50 ng mLꢀ 1), fipronil
sulfone (0, 0.5, 1.0, 2.5, 5.0, 10, and 25 ng mLꢀ 1) and fipronil sulfide (0,
1.0, 2.5, 5.0, 10, 25, and 50 ng mLꢀ 1) were obtained by diluting their
standard solution and validated by LC-MS/MS (Supplementary Table S5
– S10, Figure S10 - S13), respectively, and then analyzed with the LFI
strip and the results were visually examined in 10 min.
During detection, 100 μL fipronil standard solution or sample extract
is sprayed onto the sample pad, which flows towards the absorbent pad
along the NC membrane. In negative samples (NC, without fipronil),
GNP-mAbs were trapped by FP- ovalbumin on the T line, which turned
red. In positive samples (with fipronil), fipronil prevented GNP-mAbs
from binding with FP-ovalbumin, leading to a light red color on the T
line. The higher the concentration of fipronil in the sample, the lighter
the color of the T line. When the concentration of fipronil was sufficient
to completely block the reaction between GNP-mAbs and FP-ovalbumin,
the T line became colorless. The C line consistently turned red in
adequately assembled LFI strips. Therefore, when the C line remained
colorless, the strip was invalid or the test procedure was not adequately
performed.
2.8. Sample preparation
To prepare stock solutions, 1.0 mg fipronil or 0.1 mg fipronil me-
tabolites was dissolved in 1.0 mL acetonitrile and stored at 4 ◦C. A series
of standard solutions were prepared by directly diluting the stock so-
lution in PBS.
For accuracy and precision studies, eggs and cucumbers are pur-
chased at local supermarkets, vegetable markets and vegetable pro-
duction base in Wuxi, Jiangsu Province, respectively. The purchased
eggs and vegetables had no target pesticides residues after determina-
tion by LC-MS/MS according to the procedure reported in GB
23200.115–2018 (Li et al., 2020; Zhang et al., 2016). Subsequently, the
samples were spiked with fipronil, fipronil desulfinyl, fipronil sulfone
and fipronil sulfide, respectively. The spiked samples were used to
validate the developed icELISA and LFI strip. Samples were stored at
ꢀ 20 ◦C in low density polyethylene bags after homogenization.
For the recovery experiment of fipronil from egg liquids, 1.0 kg of
eggs were broken, and the whole liquid was collected and homogenized
for 5 min. The homogenate (10 g) was vortex-mixed with 10 mL of
fipronil acetone solution for 1 min and sonicated for 10 min at 25 ◦C. The
mixture (20 mL) was diluted five times with PBS for LFI strip analysis.
Cucumbers were ground, centrifuged, and passed through a membrane
filter. The supernatant was collected, spiked with fipronil and analyzed
using the LFI strip. The acetonitrile extract of spiked sample was diluted
by 10 times and determined by icELISA.
2.7. Sensitivity and specificity of the LFI strip
The sensitivity of the test strip was estimated by detecting different
concentrations of fipronil standard (0, 0.25, 0.5, 1.0, 2.5, 5.0, and 10 ng
mLꢀ 1) in PBS validated by LC-MS/MS (Supplementary Table S7 and S10,
Figure S11). Three replicates of each sample were analyzed. In semi-
quantitative analysis, vLOD was defined as the lowest concentration of
fipronil that resulted in a weaker color on the T line than the negative
sample. The cut-off value was defined as the threshold concentration of
fipronil that resulted in the complete disappearance of the T line (Li, Liu,
Song, Kuang, & Xu, 2018). For quantitative analysis, the relative color
intensity of the T and C lines was measured using a strip reader. A
Fig. 4. Schematic diagrams and the test principle of the LFI strip.
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J. Yao et al.
Food Chemistry 356 (2021) 129710
2.9. Matrix effects
decreased with increasing NaCl content, and acidity or alkalinity of the
assay solution contributed to comparatively higher sensitivity. Conse-
quently, 0.1% NaCl content and pH 7.4 were selected for subsequent
experiments.
The matrix effects of egg and cucumber samples were assessed. The
samples were diluted two, five, and 10 times with 0.01 mol L-1 PBS,
respectively. Matrix effects were evaluated by comparing the T lines of
egg and cucumber samples with PBS containing the same concentrations
of fipronil.
The optimized heterologous icELISA was used to analyze serial
concentrations of fipronil standard. According to the standard curve of
icELISA (Supplementary Figure S6), IC50 was 0.46 ± 0.07 ng mLꢀ 1, and
LOD was 0.05 ± 0.01 ng mLꢀ 1 with detection range from 0.05 to 2.6 ng
mLꢀ 1. Several metabolites and analogues of fipronil were analyzed.
Table S3 shows that the CRs with fipronil desulfinyl, fipronil sulfone,
and fipronil sulfide were 26%, 49%, and 20%, respectively, while the
CRs with other phenylpyrazole insecticides, such as trifluralin and
imidacioprid, were negligible. The similarity degree of molecular for-
mula between fipronil and its metabolites might contribute to the high
CRs (Supplementary Figure S1). Therefore, our developed heterologous
icELISA was highly sensitive and specific for the detection of fipronil and
its metabolites.
2.10. Accuracy and precision of the LFI strip
Accuracy and precision were assessed by detecting egg and cucum-
ber samples spiked with four fipronil concentrations. The quantitative
analysis was carried out using icELISA and the results were validated by
LC-MS/MS. On the other side, the semiquantitative analysis was ach-
ieved by LFI strip. There were three replicates per sample. The precision
of analysis method was expressed as the relative standard deviation
(RSD). Furthermore, the statistical analysis was performed using
SPSS22.0 software.
3.4. Sensitivity and specificity of the LFI strip
3. Results and discussion
According to the TEM image of the prepared GNPs (Supplementary
Figure S7), the average diameter was 25 nm. The preparation and
identification of GNP-mAbs have been previously reported (Li et al.,
2018; Peng et al., 2016; Wang et al., 2018, 2020). Subsequently, the LFI
strip was assembled as shown in Fig. 4. The color and sensitivity of the
strip is affected by pH, concentrations of mAb and coating antigen, and
GNP-mAbs resuspension through the specific binding of GNP-mAbs with
coating antigen and the chromatography. The optimum conditions of
the LFI strip consisted of pH 7.4, 5.0 × 10-3 g L-1 mAbs, 0.80 g L-1 FP-
ovalbumin, and 1.0% PVP in basic resuspension (Supplementary
Figure S8). The coating antigen was the same as that used in icELISA,
confirming that the heterologous coating antigen had high sensitivity.
Subsequently, different concentrations of fipronil standard solution
were detected using the LFI strip (Fig. 5a). It was shown that the color of
T line was gradually decreased with the increasing concentration of
fipronil, with cut-off and vLOD values of 10 and 0.25 ng mLꢀ 1, respec-
tively. The time used in this determining process was within 10 min,
indicating that the LFI strip was rapid and sensitive. Moreover, the
corresponding T lines were scanned using a strip reader, and the stan-
dard curve of quantitative immunoassay for fipronil was generated
(Fig. 5b) that T/C was plotted against the logarithm of a series con-
centration of fipronil standard solution (0–10 ng mLꢀ 1). Based on the
regression equation, IC50 was calculated to be 0.73 ± 0.04 ng mLꢀ 1, and
3.1. Identification of fipronil haptens and antigens
Considering the lack of immunogenicity, fipronil haptens need to be
synthesized for the production of anti-fipronil mAbs. As shown in
Figs. 1–3, the haptens FPA and FP were designed, synthesized, and
1
1
characterized by HPLC-MS (Fig. 2) and H NMR (Fig. 3). For FPA, H
NMR (400 MHz, DMSO‑d6) δ 8.25 ~ 8.27 (d, 2H), 8.06 (broad, 0.37),
6.80 (s, 2H). MS calculated for C12H5Cl2F6N3O3S-: [M - H+]- 453.93,
found 453.88. For FP, 1H NMR (400 MHz, DMSO‑d6) δ 8.26 (s, 2H), 8.23
(broad, 0.37H), 7.08 (s, 2H). MS calculated for C16H21N4O4S-: [M -
H+]- 362.97, found 362.96. The spectra data demonstrated that the two
haptens were successfully prepared. Immunogens and coating antigens
were produced by conjugating haptens with carrier protein and identi-
fied by UV–Vis spectrophotometry (Supplementary Figure S2). We ob-
tained a significant red shift at 240 nm with hapten FPA and the
corresponding
antigen
FPA-BSA/ovalbumin
(Supplementary
Figure S2a) and a blue shift at 210 nm with FP and FP-BSA/ovalbumin
(Supplementary Figure S2b). These results confirmed the successful
conjugation between hapten and carrier protein. Moreover, the differ-
ences of haptens FPA and FP in the structures and coupling position
might to attribute to high sensitivity of the developed heterologous
icELISA and LFI, as well as the heterology degree with the target analyte
fipronil.
LOD was 0.14 ± 0.02 ng mLꢀ 1
.
The specificity of the test strip was evaluated by analyzing fipronil
3.2. Identification of anti-fipronil mAbs
metabolites. As shown in Fig. 5c–e, the cut-off values of fipronil desul-
finyl, fipronil sulfone, and fipronil sulfide were 50, 25, and 50 ng mLꢀ 1
,
Antiserum of BALB/c mice immunized with different immunogens
was analyzed by icELISA using different coating antigens. Table S1
shows that the highest competitive inhibition was obtained in the mouse
immunized with FPA-BSA and detected by FP-ovalbumin. In addition,
heterologous ELISA had higher sensitivity than homologous ELISA. Anti-
fipronil mAbs were obtained through cell fusion, and mAbs were char-
acterized by ELISA. The mAb against fipronil from hybridoma cell line
(1B6) was an IgG2b subclass (Supplementary Figure S3). The average
affinity constant was 7.6 × 109 L molꢀ 1, and the titer was 1:1.0 × 106
(Supplementary Figure S4 and Table S2).
respectively. At 1.0 ng mLꢀ 1, the T line color was weaker for fipronil
sulfone than for fipronil desulfinyl and fipronil sulfide. The sensitivity
for fipronil metabolites was significantly lower than for fipronil, as well
as in agreement with the CRs obtained from icELISA. The results
revealed that the LFI strip could be used in the simultaneous detection of
fipronil and its metabolites.
3.5. Matrix effects
To investigate the matrix effect of eggs and cucumbers, different
dilution ratios of egg and cucumber sample stoste were tested by LFI
strip. As shown in Figure S9, the egg matrix effects were adequately
eliminated following a 5 × dilution in PBS. The matrix interference of
cucumbers was almost negligible. Given the pretreatment of egg and
cucumber samples, the pretreatment method for LFI strip was simpler
than that for instrumental analysis (Li et al., 2020; Zhang et al., 2016).
On the other hand, the results proved the high tolerance of matrix in the
developed immunoassay.
3.3. Development and optimization of icELISA
For higher sensitivity, heterologous icELISA was developed under
optimum conditions. First, a checkerboard assay was carried out to
screen the appropriate concentrations of coating antigen and mAb for
competitive assays, which were 3 × 10ꢀ 5 and 1 × 10ꢀ 5 g Lꢀ 1, respec-
tively. In addition, the effect of NaCl content and pH on ELISA was
evaluated. As shown in Supplementary Figure S5, the IC50 values
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Food Chemistry 356 (2021) 129710
Fig. 5. The semiquantitative and quantitative detection and the cross-reactivity with fipronil metabolites by LFI strip. (a) The semiquantitative assay for fipronil by
test strip. 1, 2, 3, 4, 5, 6, and 7 represent the concentrations of fipronil at 0, 0.25, 0.5, 1.0, 2.5, 5.0, and 10 ng mLꢀ 1, respectively; (b) The quantitative assay for
fipronil detection in PBS buffer by test strip; (c) The cross-reactivity with fipronil desulfinyl. 1, 2, 3, 4, 5, 6, and 7 represent the concentrations of fipronil at 0, 1.0,
2.5, 5.0, 10, 25, and 50 ng mL-1, respectively; (d) The cross-reactivity with fipronil sulfone. 1, 2, 3, 4, 5, 6, and 7 represent the concentrations of fipronil at 0, 0.5, 1.0,
2.5, 5.0, 10, and 25 ng mLꢀ 1, respectively; (e) The cross-reactivity with fipronil sulfide. 1, 2, 3, 4, 5, 6, and 7 represent the concentrations of fipronil at 0, 1.0, 2.5, 5.0,
10, 25, and 50 ng mLꢀ 1, respectively.
3.6. Accuracy and precision of the developed immunoassay for real
samples
samples spiked fipronil at 1.0–5.0
μ
g kgꢀ 1 detected by icELISA and LC-
MS/MS were not significant (P > 0.05), indicating the reliability of
the developed icELISA. When the spiked fipronil was 10
μ
g kgꢀ 1 in egg
Using the LFI strip, we analyzed egg and cucumber samples previ-
ously spiked with fipronil and its metabolites through simple pretreat-
ment. As shown in Fig. 6, the egg sample had a certain extent effect on
the color of T line and C line on LFI strip, while the sensitivity main-
tained compared with the results in Fig. 5. The protein in egg sample
might hinder the chromatography of GNPs-mAbs on LFI strip, leading to
lighter color. On the other hand, the cucumber sample had little influ-
ence on the color and sensitivity. Table S4 shows that average recoveries
of fipronil were 92.9%–98.0% by icELISA, with RSD of 2.0%–8.3%. In
accordance with the IUPCA (Thompson, Ellison, & Wood, 2002), the
RDS values were below 10% indicating the high precision of developed
immunoassay. The differences of analytical results of egg and cucumber
and cucumber samples, the averaged recoveries fell to 88% and 87%
detected by LC-MS/MS, respectively, and were acceptable (Buschmann,
2013). Whereas, the results were was significantly different with that in
acetonitrile (P < 0.05). In the semiquantitative analysis, the color of T
line gradually faded with the increase of fipronil concentration detected
by LFI strip, and the semiquantitative results were consistent with the
results obtained from icELISA and LC-MS/MS. Therefore, the LFI strip is
suitable for the rapid detection of fipronil and its metabolites in real
samples.
7

J. Yao et al.
Food Chemistry 356 (2021) 129710
Fig. 6. LFI strip for detection of fipronil and its metabolites in egg and cucumber samples. (a, e) The detection of fipronil in egg and cucumber by test strip. 1, 2, 3, 4,
and 5 represent the concentrations of fipronil at 0, 1.0, 2.5, 5.0, and 10 ng mLꢀ 1, respectively; (b, f) The detection of fipronil desulfinyl in egg and cucumber by test
strip. 1, 2, 3, 4, 5, 6 and 7 represent the concentrations of fipronil desulfinyl at 0, 2.5, 5.0, 10, 13, 25, and 50 ng mLꢀ 1, respectively; (c, g) The detection of fipronil
sulfone in egg and cucumber by test strip. 1, 2, 3, 4, 5, 6 and 7 represent the concentrations of fipronil sulfone at 0, 2.5, 5.0, 10, 13, 25, and 50 ng mLꢀ 1, respectively;
(d, h) The detection of fipronil sulfide in egg and cucumber by test strip. 1, 2, 3, 4, 5, 6 represent the concentrations of fipronil sulfide at 0, 5.0, 10, 13, 25, and 50 ng
mLꢀ 1, respectively.
4. Conclusions
Declaration of Competing Interest
A rapid and sensitive LFI strip for the detection of fipronil and its
metabolites was fabricated. Based on the CRs with fipronil desulfinyl,
fipronil sulfone, and fipronil sulfide, the LFI strip can be used for the
simultaneous detection of fipronil and its metabolites. The proposed
method paved an avenue to prepare the multi-selective antibodies to
detect chemicals in agricultural products on spot.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgment
This work is financially supported by National Key R&D Program
8

J. Yao et al.
Food Chemistry 356 (2021) 129710
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