A new residue method for the determination of flonicamid in agricultural and environmental samples using enzyme immunoassay systems

Abstract

An indirect competitive enzyme-linked immunosorbent assay (ELISA) for flonicamid was developed based on a polyclonal antibody. 4-Trifluoronicotinic acid (TFNA) was used as flonicamid hapten and conjugated to bovine serum albumin (BSA) to produce an immunogen and ovalbumin (OVA) to produce a coating antigen. Polyclonal antibodies against flonicamid were successfully produced by immunizing New Zealand white rabbits. Under the optimal conditions, the 50% inhibitory concentration (IC₅₀ value) of standard curves was 7.89 mg L⁻¹ and the limit of detection (IC₁₀) was 0.063 mg L⁻¹. There was almost no cross-reactivity of the antibody with three structurally related metabolites, indicating that the antibody had high specificity. The recoveries from spiked water, soil, tomato and apples were in the range of 82.4–113.4% with relative standard deviations of 2.9–10.3%. Moreover, the ELISA for spiked samples showed reliability and high correlation with gas chromatography. These results suggest that the proposed ELISA method has potential application for screening flonicamid in agricultural and environmental samples.

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1. Introduction

Flonicamid (N-cyanomethyl-4-trifluoromethylnicotinamide) is a novel selective systemic pyridinecarboxamide insecticide,¹ which is widely used in many countries to control aphids and other sucking insects in many crops, such as rice, fruits and vegetables because of minimal cross-resistance characteristics and lower acute mammalian toxicity.^1,2 However, the carry-over of flonicamid to agricultural products can occur and increase human exposure. To protect consumers health, sensitive and reliable analytical methods for the monitoring of flonicamid residue is of great importance.

In recent years, many methods for the determination of flonicamid residues in different types of samples have been reported, these methods include gas chromatography (GC) (Shi et al. 2015; Xie et al. 2011),^3,4 high-performance liquid chromatography (HPLC)⁵ and high-performance liquid chromatography-mass spectrometry (HPLC-MS).^1,6–9 Although these methods have high precision and sensitivity, the instruments are infeasible for large-scale and on-site analyses because they always require the long clean-up procedure and the sophisticated analytical instrumentation prior to instrumental analysis.^10,11 Therefore, there is a growing demand for simpler and more economical methods for determining flonicamid residues. Immunoassays fulfill these requirements, becoming a reliable analytical tool for screening analysis.¹²

In the study, we reported an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the analysis of flonicamid based on a polyclonal antibody. Furthermore, the accuracy and precision of the ELISA were evaluated by GC-ECD using spiked samples, and the agreement between the ELISA and GC-ECD was high.

2. Materials and methods

2.1 Materials

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 incomplete adjuvants, goat anti-rabbit IgG-horseradish peroxidase and polyoxyethylene sorbitan monolaurate (Tween-20) were purchased from Sigma Chemical Co. (Shanghai, China). All reagents and solvents were analytical grade.

2.2 Buffers and solutions

Phosphate-buffered saline (PBS, 0.01 mol L⁻¹, pH 7.4); carbonate-buffered saline (CBS, 0.05 mol L⁻¹, pH 9.6); phosphate-buffered saline containing 0.05% Tween-20 (PBST); the TMB solution contained 0.4 mmol L⁻¹ TMB and 3 mmol L⁻¹ H₂O₂ in citrate buffer (pH 5.0).

The rabbits had free access to drinking water and commercial standard laboratory diet (CZZ, Nanjing, China). They were housed according to the EEC 609/86 Directives regulating the welfare of experimental animals.

2.3 Instruments

Nuclear magnetic resonance (NMR) spectrum was recorded on a DRX 500 spectrometer (Bruker, Germany). Mass spectral (MS) data was obtained with a LC-MS^QDECA (Finigan, USA). Absorbances were read with an Infinite M200 microtiter plate reader (Tecan, Switzerland) at 450 nm, and the ELISA plates were washed with a Wellwash Plus (Thermo, USA). The antibody was freeze-dried using an Allegra 64R centrifuge (Beckman Coulter, Inc., Brea, CA). The flonicamid ELISA was confirmed with an Agilent 7890A gas chromatograph (Agilent, USA).

2.4 Preparation of immunogen and coating antigen

The structures for flonicamid and 4-trifluoronicotinic acid (TFNA) are shown in Fig. 1. TFNA used as flonicamid hapten, was conjugated to BSA using the active ester method and to OVA via the mixed anhydride method as described in the literature.¹² The conjugates were dialyzed against PBS for 72 h at 4 °C and stored at −20 °C. TFNA–BSA conjugate was used as immunogen for antibody preparation, while the TFNA–OVA conjugate served as coating antigen for ELISA establishment. The number of hapten molecules per molecule of protein (hapten density) of conjugate was estimated directly by the molar absorbance at 280 nm.

Hapten density = (ε_conjugation − ε_protein)/ε_hapten

Fig. 1 Molecular structures for flonicamid and TFNA.

2.5 Immunization and antibody preparation

Two male adult New Zealand rabbits were immunized with TFNA–BSA to raise polyclonal antibodies according to the method described previously.¹³ The first injection was 2 mg of immunogen diluted in physiological saline and emulsified with Freund's complete adjuvant. The emulsion was then injected at multiple sites on each rabbit's back. Three weeks after the first injection, 3 mg of immunogen with Freund's incomplete adjuvant was injected as a booster shot, which was given four times at 2 week intervals. After the last injection, the titres of two rabbits were more than 1 : 100 000. The antiserum was isolated from the blood by centrifugation and purified by precipitation with caprylic acid–ammonium sulphate and stored at −20 °C after freeze-drying.¹⁴

2.6 Coating of the microplate wells

Microplates were coated overnight at 4 °C with 100 μL per well of the TFNA–OVA diluted in CBS. The plates were then washed five times with PBST before the blocking buffer (5% skim milk in PBS, 200 μL) was added to each well. After another washing step, the plates were stored at 4 °C in sealed packages.

2.7 ELISA protocol

Standards or samples (50 μL per well) were added to the coated wells, followed by the addition of antibody diluted in assay buffer (50 μL per well). After incubation for 1 h at 37 °C, the plates were washed. Subsequently, 100 μL per well of goat anti-mouse IgG-horseradish peroxidase diluted in PBST was added and incubated for 1 h at 37 °C, and after another washing step, 100 μL per well of TMB solution was added and incubated for 15 min at 37 °C. Finally, 2 mol per well of sulfuric acid (50 μL per well) was added and the absorbance was measured at a wavelength of 450 nm. The measurement was run three times in triplicate wells.

2.8 Cross-reactivity

The specificity of polyclonal antibodies was determined by cross-reactivity (CR) values. CR was studied using standard solutions of flonicamid and some of its metabolites. CR values were calculated as follows:

CR% = (IC₅₀ of flonicamid/IC₅₀ of its metabolites) × 100.

2.9 Analysis of spiked samples

A total of six samples including two water samples (pond water and rice field water), two soil samples (vegetable field soil and rice field soil) and two food samples (tomato and apples) were collected from the suburb and local supermarket of Zhenjiang (China). Each analysis was performed in triplicate.

Pond water and rice field water samples were filtered and spiked with flonicamid standards at 0.1, 1 and 10 mg L⁻¹. The spiked water samples were directly analyzed by ELISA to estimate the recoveries.

Before spiking, soil samples were dried, homogenized and sieved, while vegetable and fruit samples were finely chopped and grinded. Soil, tomato and apples samples (10 g of each sample) were spiked with flonicamid at various levels (1–10 mg kg⁻¹), and 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 vigorously shaken. The organic phase (2 mL) was evaporated in vacuo. The residue was dissolved in PBS containing 10% methanol, and then analyzed by ELISA. The recoveries and relative standard deviation (RSD) were calculated.

In order to evaluate the correlation between the ELISA and GC, the above spiked samples were analyzed by GC. For GC method, the residues of flonicamid were extracted from spiked samples with acetonitrile and water according to ELISA procedure described above, and purified through an aminopropyl solid-phase extraction column. After the eluate collected was concentrated under vacuum, the samples were diluted with acetone and detected by GC. The limit of quantitation of GC for flonicamid was 0.02 mg kg⁻¹. The measured results were compared with the ELISA results.

The GC-electron capture detector (ECD) analysis was performed on a DB-1701-capillary column (30 m × 320 μm × 0.25 μm). The column temperatures were 80 °C for 1 min, a temperature increase to 240 °C at 15 °C min⁻¹ and hold for 1 min, then raised to 260 °C at 10 °C min⁻¹ and held for 10 min; a carrier gas (N₂) flow rate of 2.5 mL min⁻¹; an injection temperature of 260 °C using the splitless mode.⁴

3. Results and discussions

3.1 Identification of conjugations

In the UV-Vis spectra obtained from continuous wavelength scanning, there were a lot of obvious differences between the conjugate and the corresponding carrier protein (Fig. S1†). The characteristic peak for TFNA–BSA showed a blue-shift at 267 nm compared with the 278 nm for BSA, indicating the successful conjugation between TFNA and BSA. The coating antigen TFNA–OVA gave a UV spectrum similar to that of TFNA–BSA. The molar ratios were estimated as 23 : 1 and 8 : 1 for TFNA–BSA and TFNA–OVA, respectively.

3.2 Optimization of ELISA conditions

By the checkerboard titration method, when the maximum of absorbance (A_max) value of the ELISA reached 1.0, the corresponding concentration of coating antigen (0.21 μg mL⁻¹) and antibody (2.5 ng mL⁻¹) were selected as ELISA operation concentration.

To improve the sensitivity of the ELISA, several experimental factors, such as the salt concentration, pH and solvents, were studied. The IC₅₀ value and A_max/IC₅₀ ratio were used as the primary criteria to evaluate the ELISA performance, the lowest IC₅₀ value and the highest ratio of A_max/IC₅₀ indicated the highest sensitivity.¹⁵

Because the flonicamid is lipophilic, and methanol is common solvent used in immunoassay to improve analyte solubility.¹⁶ So, methanol was selected to improve solubility of analytes and evaluate its effect on the ELISA of flonicamid. As shown in Fig. 2a, the IC₅₀ value tended to increase with the increase of the concentrations of methanol, while the A_max/IC₅₀ showed a drastic decrease above 10% methanol. When the concentration of methanol was at 10%, ELISA showed the highest A_max/IC₅₀ and the lowest IC₅₀. The ionic strength also influenced ELISA performance (Fig. 2b). With the increase of Na⁺, the ratios of A_max/IC₅₀ increased first and then decreased, while the IC₅₀ value was on the contrary. The highest A_max/IC₅₀ and the lowest IC₅₀ were obtained at 0.4 mol L⁻¹ Na⁺. No significant effect upon the IC₅₀ and A_max/IC₅₀ was detected at pH values ranging from 4.5 to 9.5 (Fig. 2c). With a pH close to neutral, the assay was more sensitive. So, pH 7.4 was selected for the ELISA.

Fig. 2 Effect of organic solvent, ionic strength, and pH value on ELISAs.

In this study, the optimal conditions (10% methanol, pH 7.4, and an ionic strength of 0.4 mol L⁻¹) were used for the subsequent assays.

3.3 Sensitivity and specificity of the assay

Under optimal assay conditions, the ELISA standard curve for flonicamid detection was constructed (Fig. 3). A limit of detection (LOD, IC₁₀) and the sensitivity (IC₅₀) of the ELISA were 0.063 mg L⁻¹ and 7.89 mg L⁻¹, respectively. The linear working range determined as the concentrations causing 10–90% inhibition, was 0.063–887.2 mg L⁻¹ for the ELISA.

Fig. 3 Standard curve for flonicamid by ELISA.

Compared to the maximum residue limits (MRLs) of flonicamid (0.45 mg kg⁻¹ for carrot in the USA, 0.4 mg kg⁻¹ for tomato and 1 mg kg⁻¹ for apple in Japan), the sensitivity of the ELISA can meet the requirements of detection of flonicamid. The LOD values of GC and LC-MS/MS were 0.01 mg kg⁻¹ and 0.0025 mg L⁻¹, respectively.^1,4 Although the methods have high sensitivity, the instruments are expensive and not feasible for large-scale and on-site analyses. The pre-treatment for ELISA analysis finishes only extraction and dilution of sample extracts, whereas that for the instrumental methods requires the long clean-up procedure and the sophisticated analytical instrumentation. Thus, the ELISA is a simpler and more economical tool for the monitoring of flonicamid compared with the conventional methods.

The values of cross-reactivity of the antibody with flonicamid and its metabolites are showed in Table 1. There was almost no recognition of antibody with three other metabolites (the CR was <0.08%). The results clearly demonstrated that the polyclonal antibodies obtained in this study exhibited very high specificity to the target analyte flonicamid.

Table 1 Cross-reactivity of flonicamid and its analogs

Compound	Structure	IC50 (mg L^−1)	CR (%)
Flonicamid		7.89	100
TFNA–AM		98	0.08
TFNA		>1000	<0.01
TFNG		>1000	<0.01

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3.4 Analysis of spiked samples

Table 2 shows the recoveries and RSD of the tested samples. Acceptable recoveries of 82.4–113.4% and RSD of 2.9–10.3% were obtained. These data are well within the requirements of residue analysis, and the results indicated that the optimized ELISA was a potential screening tool for flonicamid residues.

Table 2 Recovery of flonicamid in spiked agricultural and environmental samples

Sample	Spiked concentration (mg L^−1, mg kg^−1)	Dilution times	Mean recovery ± SD (%, n = 3)	RSD (%)
Rice paddy water	10	0	98.5 ± 6.5	6.6
	1		93.3 ± 7.3	7.8
	0.1		89.7 ± 7.6	8.5
Pond water	10	0	85.9 ± 6.3	7.3
	1		89.3 ± 5.5	6.2
	0.1		88.1 ± 4.3	4.9
Rice paddy soil	10	2	95.1 ± 7.1	7.5
	5		96.3 ± 2.8	2.9
	1		101.4 ± 6.8	6.7
Vegetable field soil	10	2	88.0 ± 9.1	10.3
	5		87.4 ± 6.5	7.4
	1		99.5 ± 4.9	4.9
Tomato	10	2	113.4 ± 6.9	6.1
	5		103.4 ± 3.7	3.6
	1		98.5 ± 5.8	5.9
Apple	10	2	87.5 ± 6.5	7.4
	5		82.4 ± 5.4	6.6
	1		89.5 ± 3.6	4.0

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3.5 Validation of the assay with GC-ECD

To validate the applicability of the ELISA, three types of spiked samples (two water samples, two soil samples and two agricultural samples) were pretreated as described above and measured by GC-ECD and ELISA simultaneously. The results are given in Fig. 4, a good correlation between GC(x) and ELISA(y) was obtained with the linear regression equation y = 1.06x − 0.01 (R² = 0.9956, n = 18).

Fig. 4 Correlation between ELISA and GC for the spiked samples.

The analytical characteristics of the proposed ELISA with that of the GC were weighed. For GC method, the sample pre-treatment requires 2–3 h. On the other hand, since the pre-treatment of the ELISA only need extraction and dilution steps, about 50 min are required per one sample. Moreover, when a large number of samples are determined, the ELISA method is more efficient. The paper of Watanabe et al.^17,18 have a similar conclusion: “the ELISA is possible to cut in approximately 90% on required time for acquirement of analytical results when 40 samples are simultaneously handled as an example.”

4. Conclusion

In summary, an indirect competitive ELISA for flonicamid residue was successfully developed based on a polyclonal antibody. The antibody showed high specificity and the cross-reactivity for its metabolites was below 0.08%. Several spiked agricultural and environmental samples were analyzed by ELISA, and the accuracy and precision were satisfied with the requirements for residue analysis. A good correlation between the ELISA and GC results was obtained from the spiked samples. The proposed ELISA method shows the advantages of simple, economical, and high sample throughput, displaying potential application for flonicamid analysis.

Acknowledgements

This work was supported by the Natural Science Foundation of Jiangsu Province (BK20140543), the China Postdoctoral Science Foundation (2014M561596), the National Natural Science Foundation of China (31170386, 31570414), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Jiangsu Collaborative Innovation Center of Technology and Material of Water Treatment.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra27425g

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