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Ultrasensitive quantitation of imidacloprid in vegetables by colloidal gold and time-resolved fluorescent nanobead traced lateral flow immunoassays
Food Chemistry 311 (2020) 126055
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Ultrasensitive quantitation of imidacloprid in vegetables by colloidal gold and time-resolved fluorescent nanobead traced lateral flow immunoassays
T
Guiyu Tana,b, Yajie Zhaoa, Mian Wanga, Xiaojiao Chena, Baomin Wanga, , Qing X. Lic ⁎
a
College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, China Guangxi Botanical Garden of Medicinal Plants, Nanning 530023, China c Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822, United States b
ARTICLE INFO
ABSTRACT
Keywords: Lateral flow immunoassay Colloidal gold Time-resolved fluorescent Imidacloprid
Imidacloprid is a highly effective insecticide, but its potential hazards to the environment and ecosystems have limited its use in many regions. A fast and sensitive analytical method would aid monitoring imidacloprid residues. The monoclonal antibody 4D9, colloidal gold and time-resolved fluorescent nanobeads (CGN and TRFN, respectively) were applied to develop a lateral flow immunoassay (LFIA) for imidacloprid detection in the present work. Both the optimized TRFN-LFIA and CGN-LFIA had a similar limit of detection of approximately 0.02 ng/mL. Use of the portable optical scanner with the TRFN-LFIA or CGN-LFIA could quantitate concentrations of imidacloprid in vegetable as sensitively as the enzyme-linked immunosorbent assay (ELISA). The LFIAs quantitative results of imidacloprid in commercial Chinese leeks were verified by the liquid chromatography-mass spectrometry with a R2 of 0.91. The LFIAs are portable and simple and thus could fully replace the ELISA for onsite quantitation of imidacloprid residues in vegetables.
1. Introduction Imidacloprid (IMI) is a systemic neonicotinoid insecticide used to control termites, turf insects, ectoparasites, and agricultural pests. It inhibits nicotinic acetylcholine receptors in the insect central nerve system (Bai, Lummis, Leicht, Breer, & Sattelle, 2010; Buckingham, Lapied, Corronc, & Sattelle, 1997; Liu, Lanford, & Casida, 1993). Due to its widespread use, a large amount of IMI has entered the environment and has been accumulated in agricultural products, which would eventually pose a threat to human health. Since 2013, the European Union (EU) had strictly restricted the use of three neonicotinoids including IMI. The entire EU then in April 2018 banned the use of IMI in open areas. After a long-time debate, the sale and use of pesticides containing neonicotinoids had been banned in France since September 2018, because the beekeepers had noticed a significantly increased mortality rate in honeybee due to the heavy use of neonicotinoid insecticides. In Canada, high levels of IMI were detected in water near high-density plantations, which led to the Canadian Health Ministry plan to phase out the use of IMI in all agricultural products. It is of, therefore, significance to monitor IMI in agricultural products. Currently, instrument methods are often used for the accurate detection of IMI in samples, including gas chromatography-mass spectrometry (GC–MS) (Lodevico & Li, 2002), high performance liquid ⁎
chromatography (HPLC) (Rancan, Sabatini, Achilli, & Galletti, 2006; Rossi, Sabatini, Cenciarini, Ghini, & Girotti, 2005), and HPLC-mass spectrometry (HPLC-MS) (Hengel & Miller, 2008; Takashi, Tomomi, & Eiki, 2014). Such methods have the advantages of high accuracy and reproducibility, however, they require highly skilled personnel, tedious sample pretreatment, and are not convenient for on-site detection. Immunoassay is an analytical method based on the interaction between an antibody and the corresponding antigen, which have been recruited to detect IMI. The first immunoassay for IMI was reported by Li and Li (2000). They synthesized an outstanding hapten and most of the following antibodies against IMI were developed with the same hapten. The maximum half inhibition concentration (IC50) of the established enzyme-linked immunosorbent assays (ELISAs) ranged from 0.3 to 6.8 ng/mL (Tan, Chen, & Liu, 2018). Furthermore, the bare-eye based semi-quantitation lateral flow immunoassays (LFIAs) using the same hapten were established by Xu et al. (2012) and Fang et al. (2015). The quantum-dot-based LFIA was also applied to broad-specific detection of neonicotinoids including IMI, imidaclothiz, and clothianidin (Wu et al., 2018). The LFIA is a popular first-level screening tool. It has been wellknown for the attractive property of rapid and easy operation, economical and sensitive analysis (Anfossi, Baggiani, Giovannoli, & Arco, & Giraudi, 2013; Dzantiev, Byzova, Urusov, & Zherdev, 2014). However,
Corresponding author. E-mail address: [email protected] (B. Wang).
https://doi.org/10.1016/j.foodchem.2019.126055 Received 26 June 2019; Received in revised form 19 November 2019; Accepted 11 December 2019 Available online 14 December 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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the LFIA also have shortcomings, including lack of automated documentation, subjective interpretation of results, as well as inaccurate quantification (Posthuma-Trumpie, Jakob, & Aart, 2009). To overcome the abovementioned limitations, both handheld and portable LFIA readers were developed in recent years, which fulfilled the features of on-site quantitative analysis. For commercial LFIA, the colloidal gold nanobead (CGN) is the most commonly used label. It is inexpensive to prepare and can be visualized for quantitation (Mak, Beni, & Turner, 2016). On the contrary, the time-resolved fluorescent nanobead (TRFN), lanthanide Eu(III) labeled, applied in the LFIA has been increasingly reported in recent years. It possesses very large Stoke’s shifts and much longer decay times than traditional fluorescent reagents, and distinguished the useful signal from the nonspecific fluorescence signal. Therefore, it has been considered as a new quantitative screening tool for the target analyst (Goryacheva, Lenain, & De Saeger, 2013; Qi, Qu, Chen, Liu, & Li, 2017). A large number of research papers had appeared in recent years describing the development of LFIA with TRFN probe, of which the sensitivity improved greatly in comparison with the CGNLFIA (Fu, Chu, Zhao, Li, & Deng, 2017; Liu et al., 2012; Wang et al., 2015; Zhang, Li, Zhang, & Zhang, 2011; Zhang et al., 2009). In the present study, based on an ultrasensitive mAb to IMI, applying CGN and TRFN tracers for optical signal output, we developed two LFIAs for quantitation of IMI residues. The sensitivity, specificity and reproducibility of the two LFIAs were assessed and compared under an optimum condition. The suitability of LFIAs for on-site quantitation of IMI in vegetables was evaluated with a simple extract method and the portable optical readers.
2.3. Preparation of antibody probes
2. Materials and methods
The NC membrane was attached to the center of the PVC plate. Goat anti-mouse IgG (Control line, C line) and IMI-BSA (Test line, T line) were immobilized on the NC membrane at 1 μL/cm and separated by a distance of 0.5 cm. The conjugate pad was saturated with the probemAb conjugate and the sample pad was pasted on the downstream of the NC membrane sequentially. The absorption pad was pasted on the upstream. Each pad was pasted with a 0.2 cm overlap with the adjacent pad. The individual strips were placed in plastic cases, sealed in aluminum foil, and stored at 4 °C.
2.3.1. Preparation of CGN-mAb probe CGN particle (30 nm) was adjusted to pH 7.8 with potassium carbonate solution. The mAb aqueous solution was then added to the CGN solution (6 µg/mL) and stirred for 10 min. The coated CGN-mAb suspension was stabilized by adding 10% (w/v) BSA and stirred for a further 10 min, followed by centrifugation at 10,000 rpm for 10 min. The supernatant was discarded, and the CGN-mAb conjugate was resolved with Na2HPO4-KH2PO4 buffer (pH 7.4, 0.01 M) to obtain a 1% volume of the original CGN solution. 2.3.2. Preparation of TRFN-mAb probe To activate the nanobeads, 1 mL of Eu(III)-nanobeads was added to 9 mL of ddH2O, followed by adding 0.4 mL of fresh 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide solution (50 mg/mL, dissolved by ethanol respectively) to the mixture. The mixture was shaken for 30 min at room temperature and then centrifuged at 10,000 rpm for 20 min. The precipitate was suspended in 10 mL of ddH2O by ultrasonication. Next, 1 mL of mAb (1 mg/mL) was added dropwise to the activated nanobead for conjugation. The solution was shaken for 1 h at room temperature and then blocked with 10% BSA for another 1 h. The mixture was centrifuged at 10,000 rpm for 20 min and the precipitate was re-suspended in 5 mL of boric acid buffer (0.05 M, pH 8.5) containing 0.5% Tween-20 and 0.2% BSA. 2.4. Fabrication of strip
2.1. Materials and equipment The IMI standard and its analogues, bovine serum albumin (BSA), goat anti-mouse IgG, and PEG 2000 were purchased from Sigma (Shanghai, China). CGN (0.01% , w/v), nitrocellulose (NC) membranes (Sartorius CN140), sample pads, absorbent pads, polyvinyl chloride (PVC) plate and plastic cases were purchased from Jieyi Biotech Co., Ltd. (Shanghai, China). TRFN (1%, w/v) and a Microdetection® fluorescent scanner were purchased from Nanjing Microdetection Biotech Co., Ltd. (Nanjing, China). The colloidal gold scanner was purchased from Shanghai Jiening Biotech Co., Ltd (Shanghai, China). All other reagents were purchased from Beijing Chemical Reagents Co. (Beijing, China).
2.5. Optimization of LFIA Checkerboard titrations were conducted with various concentrations of IMI-BSA and probe-mAb to achieve the best sensitivity and detectability of signals. The kinetic analysis of the LFIAs was applied to obtain a best reaction time for repeatable quantitative analysis. The solvent effect with the organic solvents was optimized for the loading buffer.
2.2. Development of monoclonal antibody against IMI The hapten II of IMI from our previous work (Li & Li, 2000) was conjugated with BSA (IMI-BSA) to obtain an immunogen as previously reported (Li & Li, 2000; Lee, Ahn, Park, Kang, & Hammock, 2001). The monoclonal antibody (mAb) against IMI was produced using the hybridoma antibody technology (Guo et al., 2016). Briefly, a Bal b/c mouse was injected with immunogen for four times, and then the spleen cell was applied for fusion with murine SP 2/0 myeloma cells via PEG 2000. With an ELISA screening procedure, the monoclonal hybridoma cell line was selected to prepare ascites, and the anti-IMI mAb was further purified by the ammonium sulfate technique and stored at –40 °C before use. The animal experimental was performed strictly according to the standards described in the Guide for the Care and Use of Laboratory Animals (National Research Council Commission on Life Sciences, 1996 edition). All the animal treatment procedures were approved by the Animal Care Committee of China Agricultural University.
2.5.1. Optimization the concentration of probe-mAb on the conjugation pad The CGN-mAb conjugate solution was diluted 20-, 30-, 40-, and 50fold with PBS and then sprayed onto the conjugate pad, respectively. For the TRFN-mAb conjugate solution, the dilutions of 20-, 30-, 40-, and 50-fold with boric acid buffer (0.05 M, pH 8.5) were prepared, and then sprayed onto the conjugate pad. 2.5.2. Optimization the concentration of IMI-BSA on the T line The IMI-BSA at concentrations of 0.6, 0.8 and 1.0 mg/mL were immobilized on the T line. The C line was immobilized with 0.5 mg/mL of goat anti-mouse IgG. The NC membrane was then combined with the conjugate pad with serial contents of probe-mAb to construct strips. A volume of 100 µL PBS containing 0 ng/mL and 1 ng/mL IMI standard was pipetted into the sample well of the LFIAs. The optical signals of the T line and C line of the LFIAs were read and recorded by a stripe reader. 2.5.3. Optimization of the reaction time An aliquot of 100 µL of PBS containing 0 ng/mL and 1 ng/mL IMI 2
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standard was pipetted into the sample well of the LFIAs. After 2 min of incubation, the signal intensity of the LFIAs was read by the strip reader every 2 min. The signal intensity ratio of T line to C line against time was plotted to establish the kinetic curves. Each measurement was performed in triplicate.
filtered through 0.22 μm filter prior to analysis. 2.7.3. LC-MS/MS analysis A Shim-pack XR-ODS III column (150 mm × 2.0 mm, i.d., 2.2 μm; Shimadzu Corp., Japan) was applied in the column oven kept at 40 °C. The injection volume was 1 μL. The mobile phase with a flow rate of 0.4 mL/min consisted of eluent A (methanol) and eluent B (1 mmol/L aqueous ammonium acetate). The mobile phase was started with 10% A and 90% B, The proportion of A linearly increased to 50% over 4 min, and then to 75% over 16 min. The proportion of A was increased to 95% over 2 min and held for 5 min, then immediately adjusted to its initial composition and held for 8 min. The retention time of IMI was 5.27 min. For MS analysis, the electrospray ionization source was operated in both positive and negative mode (4,000 V). High-purity N2 served both as sheath and auxiliary gas. Argon (2.7 × 105 Pa) was used as the collision-induced dissociation gas. Detection was carried out in the MRM mode. The mass spectroscopy transitions and fragmentation conditions selected for IMI are m/z 256.1 → 175.1 at −16 eV.
2.5.4. Optimization of the loading buffer Methanol and acetonitrile were often used to extract IMI from matrices. Effects of methanol and acetonitrile were studied using PBS containing 0, 5, and 10% of the organic solvents to dilute the IMI standard. These solutions were pipetted into the sample well of the LFIAs. The optical signals of the T line and C line were recorded. Each measurement was performed in triplicate. 2.6. Evaluation of the LFIA 2.6.1. Standard curves A stock solution of IMI was diluted with PBS to a series of concentrations for standard solutions. Each solution was added into the sample well of LFIA. After incubation, the optical intensity ratio of the T line to the C line was read for each concentration as B. The standard curve was obtained by plotting the ratio of the B value to the B0 value (T/C of the 0 ng/mL) versus the logarithm of IMI concentration, and they were fitted into a four-parameter logarithmic equation. The IC50 value was taken as the assay sensitivity. The linear working range was represented by IC20-IC80 (Van Emon, 2007). The IC10 was used to calculate the limit of detection (LOD) as previously reported (Brady, 1995). Each measurement was performed in triplicate. The standard curve generation and data treatment were performed with OriginPro 8.1 (OriginLab).
3. Results and discussion 3.1. Development of monoclonal antibody against IMI With the hybridoma antibody technology, we obtained the anti-IMI mAbs. The best clone, designated as 4D9, was used to produce ascites. The ascites titer was 8 × 105 and further purified by the ammonium sulfate technique. Using the mAb 4D9, an inhibition curve obtained by ELISA was displayed as short dot line in Fig. 3(A). The IC50 was calculated as 0.07 ng/mL, while the dynamic range was 0.024–0.20 ng/ mL.
2.6.2. Recovery A standard recovery towards IMI was recorded to evaluate the accuracy of LFIA in vegetable samples. After determined as IMI-free samples via an LC-MS/MS method, IMI-free vegetable samples were spiked with IMI standard solution to achieve concentration gradient series. Ten microliter of these serial IMI dilutions were spiked into IMIfree samples, and then extracted and determinate by the LFIAs. The recovery was calculated as follows: Recovery (%) = (Measured amount/Spiked amount) × 100%. Each assay repeated three times.
3.2. Optimization of LFIA Checkerboard titrations were applied to optimize the concentration of IMI-BSA and probe-mAb. The results (Fig. 1) were expressed as the dilution fold of the labeled nanobeads against the T/C value. The black line and gray line displayed the inhibition trend of 0 ng/mL and 1 ng/ mL of IMI, respectively. For the CGN-LFIA (Fig. 1A), the pair, 0.8 mg/ mL of IMI-BSA and 30 × of labeled CGN-mAb, was chosen for subsequent study. Under this condition, the inhibition ratio could achieve the highest value, and it could give a minimum T/C of 1 ng/mL while maximum T/C of 0 ng/mL. For the same seasons, we chose the pair, 0.6 mg/mL of IMI-BSA and 40 × of labeled TRFN-mAb for the TRFNLFIA (Fig. 1B). To compare the two LFIAs, the CGN-LFIA was applied with approximately 2 ng of CGN for one strip, and it was approximately 0.4 ng of TRFN for the other one. While the optical signals were detected in an available range, the IMI-BSA usage of TRFN-LFIA was also less than that of CGN-LFIA, which was consistent with the previous reports (Hu et al., 2017; Pyo, 2007). The optimization of reaction time was performed by recording the optical signal every 2 min after pipetting the standard solution into the sample well. As Fig. 2 shows, both the signal intensity of CGN- and TRFN-LFIA had enhanced over time (inset graph), On the contrary, the T/C value decreased. The signal variation of 0 ng/mL was greater than that of 1 ng/mL. Both features of CGN-LFIA raised and fell more greatly than the TRFN-LFIA. Ten min after addition of the standard solution into the sample well, the optical intensity and T/C value of the TRFNLFIA were relatively constant, which enabled a long time for scanner reading. The continuous decrease of the T/C value of the CGN-LFIA showed that 10–15 min after sample addition could allow the scanner reading. The organic solvents in the immunoreaction medium often influence the analyte-antibody interaction, causing considerable interference in the assay. Thus, the effect of methanol and acetonitrile on the LFIA was evaluated in the present research. The two organic solvents in PBS at concentrations of 5% and 10% were prepared to dilute IMI for standard
2.6.3. Specificity The specificity of the LFIA for IMI was evaluated by cross-reactivity (CR). Stock solutions of clothianidin, acetamiprid, thiamethoxam, dinotefuran, and 6-chloronicotinic acid were separately prepared with PBS and pipetted to the sample well of the LFIAs. The CR was calculated according to the formula: CR (%) = (IC50 of IMI) / (IC50 of analyte) × 100. Each sample was analyzed in triplicate. 2.7. Validation of LFIA Chinese leek samples were collected from local markets, analyzed by the LFIAs and further confirmed using LC-MS/MS. Each sample was analyzed in triplicate. 2.7.1. Sample preparation for LFIA The samples were finely ground and 1 g sample was extracted in 5 mL PBS. After shaking for 10 min at room temperature, the extract was stood for 5 min, and then the supernatant was diluted with PBS before sensing. 2.7.2. Sample preparation for LC-MS/MS The sample preparation for LC-MS/MS was performed as previously described (Li, 2016). Ten grams of sample were extracted in 20 mL of acetonitrile by shaking for 30 min. Five grams of NaCl were added, followed by centrifugation at 5,000 rpm for 3 min. The supernatant was 3
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Fig. 1. Optimization of the concentration of IMI-BSA, probe-mAb (A and B) and the loading buffer (C and D).
curve (Fig. 1C and D). The two organic solvents caused mass variations of the standard curve. To avoid the effect of organic solvents, the solvents should be removed prior to the test.
3.3. Evaluation of the LFIA 3.3.1. Standard curves The standard curves of the two optimized LFIAs were constructed by plotting B/B0 against the logarithm of IMI concentration. Fig. 3 shows a
Fig. 2. Optimization of reaction time for colloidal gold lateral flow immunoassay (CGN-LFIA) and time-resolved fluorescent nanobead traced lateral flow immunoassay (TRFN-LFIA). 4
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interaction, and it is often easy and routine to perform. The TRFN and mAb were conjugated via chemical bonds. Although it is well known that chemical bond is stronger than the electrostatic interactions, the reaction for TRFN was tedious and time-consuming. Moreover, once the reaction condition was inappropriate, the precipitation appeared frequently during the reaction and was difficult to disperse. An ELISA assay was also compared with the two LFIAs. The CGNLFIA could give a full coverage of ELISA assay range. An integration of an optical scanner with the CGN-LFIA could fully replace the ELISA for one-step quantitation of IMI. 3.3.2. Recovery To test the reliability of the developed assay in practical application, Chinese leek, sweet potato and potato were fortified with IMI at concentrations of 0–40 ng/g. The average recoveries of IMI detected with CGN-LFIA were 72.3–106.3%, 69.1–111.3% and 58.6–121.4% for Chinese leek, sweet potato and potato, respectively (Table 1). The corresponding average recoveries measured with TRFN-LFIA were 78.8–108.6%, 58.1–88.7%, and 72.7–91.1%. The two major facts, extraction buffer containing no organic reagent and short extraction time, allowed the LFIA more suitable for on-site test. In addition, the spike tests showed that the IMI content below 0.5 ng/g could not be detected in vegetable samples with the LFIAs, which was far less than the detection potential. The reason might come from the simple sample preparation, which no presence of organic reagents in the extraction buffer and short extraction time allowed the LFIA more suitable for on-site tests. To improve the LOD of IMI in real samples, extract cleanup should be considered. 3.3.3. Specificity To evaluate the specificity of the LFIAs, IMI analogues (clothianidin, acetamiprid, thiamethoxam, and dinotefuran) and major metabolite 6chloronicotinic acid were used in the CR study (Table 2). The CR of clothianidin and acetamiprid were 3.6% and 1.1% via CGN-LFIA, respectively, and which were calculated as 4.1% and 1.1% using the TRFN-LFIA, respectively. There was no inhibition even at the concentration of 1000 ng/mL of thiamethoxam, dinotefuran, and 6-chloronicotinic acid. Thiamethoxam, dinotefuran, and 6-chloronicotinic acid could not disturb the assay, while clothianidin and acetamiprid only at high concentrations affected the test results.
Fig. 3. (A) Standard curves of imidacloprid detected by colloidal gold lateral flow immunoassay (CGN-LFIA), time-resolved fluorescent nanobead traced lateral flow immunoassay (TRFN-LFIA) and ELISA. (B) CGN-LFIA and (C) TRFN-LFIA strips showing color changes corresponding to concentrations of imidacloprid in PBS.
3.4. Validation of LFIA To further confirm the accuracy of the developed LFIAs, Chinese leek samples were collected and analyzed by the CGN-LFIA, TRFN-LFIA and LC-MS/MS. Table 3 shows that the LC-MS/MS data correlated well with those from LFIAs (R2, 0.91). Bland-Altman bias plots are provided in Supplemental File for comparison between the LFIA and LC-MS/MS. The IMI residue content in Chinese leek samples detected with LFIAs was between 0.7 and 50 ng/g. Samples with IMI content > 2 ng/g were verified by LC-MS/MS. The content of the sample No. 1 detected by LFIAs was 0.7 ng/g and 0.8 ng/g, which were not detected with the LCMS/MS method. The reason for this may be the sample content lower than the LOD of LC-MS/MS, or the detection result of LFIA was false positive. The commercial potato, sweet potato and most Chinese leek samples were negative when LFIAs and LC-MS/MS were used. According to the China National Food Safety Standard-Maximum Residue Limits (MRL) for Pesticides in Food (GB 2763-2016), the MRLs of IMI in vegetable range from 0.2 to 5 mg/kg. The European Commission set the MRL of 0.05 mg/kg for IMI in leek. MRLs of 0.02–5 ppm for IMI was established for the sum of the IMI residue on agricultural products by the Japan Food Chemical Research Foundation, and MRL of 0.4 ppm for IMI residue in potato vegetable was currently in effective in the United States (no MRL for leek). After unit conversion (0.7–50 ng/g equivalent to 0.7 × 10−3-0.05 mg/kg and ppm), obviously, the content of the positive leek samples presented
typical inhibition curve of the CGN-LFIA (black curve). The regression equation was Y = −0.07 + 1.07/[1+(X/0.15)0.889] with a reliable correlation coefficient R2 = 0.998. The IC50, LOD and the dynamic range were 0.13 ng/mL, 0.01 ng/mL, and between 0.028 ng/mL and 0.50 ng/mL. The inhibition curve of the TRFN-LFIA was showed as the gray curve, with an IC50 of 0.14 ng/mL, a LOD of 0.02 ng/mL, and a dynamic range of 0.039–0.54 ng/mL. The regression equation was Y = 0.05 + 0.93/[1+(X/0.13)1.165] and the correlation coefficient R2 was 0.992. Although the ELISA possessed the smallest value of IC50 among the three assays, the detection range of CGN-LFIA was larger than the other two methods. The CGN-LFIA could fully replace the ELISA when it is used to detect IMI in actual samples. Contrary to earlier reports that TRFN was capable of delivering sensitivity in a greater range (4–5 fold) than CGN (Hu et al., 2017; Pyo, 2007), there was no significant difference in the IC50 and detection range between the two LFIAs in the present study. The TRFN tracer seemed no remarkable superiority. The CGN-LFIA could achieve the same sensitivity as the TRFN-LFIA. Moreover, the LODs of the reported TRFN-LFIA assay were rare to find lower than 10 ng/L for small molecules. The conjugation of CGN and mAb relies on electrostatic 5
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Table 1 Average recoveries of imidacloprid in vegetable samples measured by colloidal gold lateral flow immunoassay (CGN-LFIA) and time-resolved fluorescent nanobead traced lateral flow immunoassay (TRFN-LFIA). Sample
Chinese leek
Sweet potato
Potato
Fortified (ng/g)
0 0.5 1 10 20 40 0 0.5 1 10 20 40 0 0.5 1 10 20 40
CGN-LFIA
TRFN-LFIA
Detected (ng/g)
Recovery (%)
SD
Detected (ng/g)
Recovery (%)
SD
ND 0.53 0.72 9.15 15.96 37.74 ND 0.56 0.69 7.13 18.14 38.81 ND 0.61 0.59 7.70 15.03 28.45
ND 106.3 72.3 90.6 79.8 94.3 ND 111.3 69.1 71.3 90.7 97.0 ND 121.4 58.6 77.0 75.1 71.1
ND 12.5 15.2 2.3 0.7 6.1 ND 28.5 1.6 0.4 4.1 7.6 ND 14.2 4.1 0.4 0.6 1.6
ND 0.46 0.82 10.86 19.13 31.52 ND 0.29 0.67 8.87 14.73 33.08 ND 0.38 0.73 8.54 14.58 36.45
ND 91.8 82.2 108.6 95.6 78.8 ND 58.1 67.3 88.7 73.6 82.7 ND 76.5 72.7 85.4 72.9 91.1
ND 3.0 9.6 2.4 3.4 1.4 ND 2.9 6.2 0.4 0.0 2.2 ND 1.4 2.3 1.7 2.3 4.1
in this paper were far less than the MRLs mentioned above. The extract of sample containing 0.02 mg/kg of IMI could be diluted 200 fold to meet the threshold value of standard curves, which ensured the elimination of matrix interference.
in real samples with the LFIAs. Further purification of sample extracts may improve the LOD. The good agreement was found between the LFIAs and LC-MS/MS for the analysis of IMI in commercial Chinese leek. The LFIAs can be used as a routine method for monitoring IMI residues in vegetables.
4. Conclusions
Author Contributions
In the present study, we developed two types of LFIAs with an ultrasensitive mAb. The assay sensitivities for IMI were greater than most reported studies and as sensitive as the ELISA. The IC50 and LOD were 0.13 and 0.01 ng/mL for CGN-LFIA, and 0.14 and 0.02 ng/mL for TRFN-LFIA, respectively. The TRFN-LFIA and CGN-LFIA had a similar LOD. The two assays had the average recovery of 58–121% for spiked IMI in vegetables. The IMI content below 0.5 ng/g could not be detected
Guiyu Tan developed the monoclonal antibody against imidacloprid, established the two lateral flow immunoassays. Yajie Zhao collected and prepared the sample and preformed the LC-MS/MS analysis. Mian Wang prepared the conjugate of hapten-carrier protein and probeantibody. Xiaojiao Chen evaluated the lateral flow immunoassays. Baomin Wang planned experiments, analysed data and supervised the
Table 2 Specificity of colloidal gold lateral flow immunoassay (CGN-LFIA) and time-resolved fluorescent nanobead traced lateral flow immunoassay (TRFN-LFIA) to imidacloprid and its analogs. Analyte
Structure
CGN-LFIA
TRFN-LFIA
IC50 (ng/mL)
Cross reactivity (%)
IC50 (ng/mL)
Cross reactivity (%)
Imidacloprid
0.13
100
0.14
100
Clothianidin
3.62
3.6
3.43
4.1
Acetamiprid
12.22
1.1
12.79
1.1
Thiamethoxam
> 5000
< 0.01
> 5000
< 0.01
Dinotefuran
> 5000
< 0.01
> 5000
< 0.01
6-Chloronicotinic acid
> 5000
< 0.01
> 5000
< 0.01
6
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Table 3 Concentrations of imidacloprid in commercial Chinese leek determined with colloidal gold lateral flow immunoassay (CGN-LFIA), time-resolved fluorescent nanobead traced lateral flow immunoassay (TRFN-LFIA) and LC-MS/MS. Sample No.
CGN-LFIA (ng/g)
TRFN-LFIA (ng/g)
LC-MS/MS (ng/g)
1 2 3 4 5 6 7 8 9 10
0.8 ± 0.2 ND ND ND 37.6 ± 2.4 56.6 ± 2.9 ND ND 8.6 ± 0.6 15.8 ± 0.0
0.7 ± 0.2 ND ND ND 27.5 ± 3.2 41.3 ± 2.6 ND ND 9.2 ± 1.2 13.4 ± 0.5
ND ND ND ND 28.6 ± 1.1 47.7 ± 0.8 ND ND 7.8 ± 0.4 14.6 ± 0.8
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G., Achilli, G., & Galletti, G. C. (2006). Determination of Imidacloprid and metabolites by liquid chromatography with an electrochemical detector and post column photochemical reactor. Analytical Chimica Acta, 555(1), 20–24. Rossi, S., Sabatini, A. G., Cenciarini, R., Ghini, S., & Girotti, S. (2005). Use of high-performance liquid chromatography–uv and gas chromatography-mass spectrometry for determination of the imidacloprid content of honeybees, pollen, paper filters, grass, and flowers. Chromatographia, 61(3–4), 189–195. Takashi, I., Tomomi, O., & Eiki, W. (2014). Water-based extraction and liquid chromatography-tandem mass spectrometry analysis of neonicotinoid insecticides and their metabolites in green pepper/tomato samples. Journal of Agricultural and Food Chemistry, 62(13), 2790. Tan, G. Y., Chen, J. Y., & Liu, W. H. (2018). Study progress on determination of pesticide imidacloprid residue by enzyme-linked immunoassay. Journal of Anhui Agricultural Sciences, 46(12), 17–19. Van Emon, J. M. (2007). Immunoassay and other bioanalytical techniques. CRC Press/Taylor & Francis. Wang, D., Zhang, Z., Li, P., Zhang, Q., Ding, X., & Zhang, W. (2015). Europium nanospheres-based time-resolved fluorescence for rapid and ultrasensitive determination of total aflatoxin in feed. Journal of Agricultural and Food Chemistry, 63(47), 10313–10318. Wu, S., Liu, L., Duan, N., Li, Q., Zhou, Y., & Wang, Z. (2018). An aptamer-based lateral flow test strip for rapid detection of zearalenone in corn samples. Journal of Agricultural and Food Chemistry, 66(8), 1949–1954. Xu, T., Xu, Q. G., Li, H., Wang, J., Li, Q. X., Shelver, W. L., & Li, J. (2012). Strip-based immunoassay for the simultaneous detection of the neonicotinoid insecticides imidacloprid and thiamethoxam in agricultural products. Talanta, 101, 85–90. Zhang, D., Li, P., Zhang, Q., & Zhang, W. (2011). Ultrasensitive nanogold probe-based immunochromatographic assay for simultaneous detection of total aflatoxins in peanuts. 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project. Qing X. Li prepared the hapten. Guiyu Tan, Baomin Wang and Qing X. Li wrote the manuscript with contributions from all other authors. Funding This work was supported by the National Key Research and Development Program of China [grant number 2018YFC1602900] and the Guangxi Science and Technology Major Project [grant number AA17204043]. Declaration of Competing Interest 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. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.126055. References Anfossi, L., Baggiani, C., Giovannoli, C., D‘Arco, G., & Giraudi, G. (2013). Lateral-flow immunoassays for mycotoxins and phycotoxins: A review. Analytical & Bioanalytical Chemistry, 405(2–3), 467–480. Bai, D., Lummis, S. C. R., Leicht, W., Breer, H., & Sattelle, D. B. (2010). Actions of imidacloprid and a related nitromethylene on cholinergic receptors of an identified insect motor neurone. Pest Management Science, 33(2), 197–204. Brady, J. F. (1995). Interpretation of immunoassay data. Acs Symposium, 266–287. Buckingham, S., Lapied, B., Corronc, H., & Sattelle, F. (1997). Imidacloprid actions on insect neuronal acetylcholine receptors. Journal of Experimental Biology, 200(Pt 21), 2685–2692. Dzantiev, B. B., Byzova, N. A., Urusov, A. E., & Zherdev, A. V. (2014). Immunochromatographic methods in food analysis. Trends in Analytical Chemistry, 55(55), 81–93. Fang, Q., Wang, L., Cheng, Q., Cai, J., Wang, Y., Yang, M., ... Liu, F. (2015). A bare-eye based one-step signal amplified semiquantitative immunochromatographic assay for the detection of imidacloprid in Chinese cabbage samples. Analytical Chimica Acta, 881, 82–89. Fu, X., Chu, Y., Zhao, K., Li, J., & Deng, A. (2017). Ultrasensitive detection of the βadrenergic agonist brombuterol by a SERS-based lateral flow immunochromatographic assay using flower-like gold-silver core-shell nanoparticles. Microchimica Acta, 184(6), 1711–1719. Goryacheva, I. Y., Lenain, P., & De Saeger, S. (2013). Nanosized labels for rapid immunotests. Trends in Analytical Chemistry, 46, 30–43.
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Ultrasensitive quantitation of imidacloprid in vegetables by colloidal gold and time-resolved fluorescent nanobead traced lateral flow immunoassays
T
Guiyu Tana,b, Yajie Zhaoa, Mian Wanga, Xiaojiao Chena, Baomin Wanga, , Qing X. Lic ⁎
a
College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, China Guangxi Botanical Garden of Medicinal Plants, Nanning 530023, China c Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822, United States b
ARTICLE INFO
ABSTRACT
Keywords: Lateral flow immunoassay Colloidal gold Time-resolved fluorescent Imidacloprid
Imidacloprid is a highly effective insecticide, but its potential hazards to the environment and ecosystems have limited its use in many regions. A fast and sensitive analytical method would aid monitoring imidacloprid residues. The monoclonal antibody 4D9, colloidal gold and time-resolved fluorescent nanobeads (CGN and TRFN, respectively) were applied to develop a lateral flow immunoassay (LFIA) for imidacloprid detection in the present work. Both the optimized TRFN-LFIA and CGN-LFIA had a similar limit of detection of approximately 0.02 ng/mL. Use of the portable optical scanner with the TRFN-LFIA or CGN-LFIA could quantitate concentrations of imidacloprid in vegetable as sensitively as the enzyme-linked immunosorbent assay (ELISA). The LFIAs quantitative results of imidacloprid in commercial Chinese leeks were verified by the liquid chromatography-mass spectrometry with a R2 of 0.91. The LFIAs are portable and simple and thus could fully replace the ELISA for onsite quantitation of imidacloprid residues in vegetables.
1. Introduction Imidacloprid (IMI) is a systemic neonicotinoid insecticide used to control termites, turf insects, ectoparasites, and agricultural pests. It inhibits nicotinic acetylcholine receptors in the insect central nerve system (Bai, Lummis, Leicht, Breer, & Sattelle, 2010; Buckingham, Lapied, Corronc, & Sattelle, 1997; Liu, Lanford, & Casida, 1993). Due to its widespread use, a large amount of IMI has entered the environment and has been accumulated in agricultural products, which would eventually pose a threat to human health. Since 2013, the European Union (EU) had strictly restricted the use of three neonicotinoids including IMI. The entire EU then in April 2018 banned the use of IMI in open areas. After a long-time debate, the sale and use of pesticides containing neonicotinoids had been banned in France since September 2018, because the beekeepers had noticed a significantly increased mortality rate in honeybee due to the heavy use of neonicotinoid insecticides. In Canada, high levels of IMI were detected in water near high-density plantations, which led to the Canadian Health Ministry plan to phase out the use of IMI in all agricultural products. It is of, therefore, significance to monitor IMI in agricultural products. Currently, instrument methods are often used for the accurate detection of IMI in samples, including gas chromatography-mass spectrometry (GC–MS) (Lodevico & Li, 2002), high performance liquid ⁎
chromatography (HPLC) (Rancan, Sabatini, Achilli, & Galletti, 2006; Rossi, Sabatini, Cenciarini, Ghini, & Girotti, 2005), and HPLC-mass spectrometry (HPLC-MS) (Hengel & Miller, 2008; Takashi, Tomomi, & Eiki, 2014). Such methods have the advantages of high accuracy and reproducibility, however, they require highly skilled personnel, tedious sample pretreatment, and are not convenient for on-site detection. Immunoassay is an analytical method based on the interaction between an antibody and the corresponding antigen, which have been recruited to detect IMI. The first immunoassay for IMI was reported by Li and Li (2000). They synthesized an outstanding hapten and most of the following antibodies against IMI were developed with the same hapten. The maximum half inhibition concentration (IC50) of the established enzyme-linked immunosorbent assays (ELISAs) ranged from 0.3 to 6.8 ng/mL (Tan, Chen, & Liu, 2018). Furthermore, the bare-eye based semi-quantitation lateral flow immunoassays (LFIAs) using the same hapten were established by Xu et al. (2012) and Fang et al. (2015). The quantum-dot-based LFIA was also applied to broad-specific detection of neonicotinoids including IMI, imidaclothiz, and clothianidin (Wu et al., 2018). The LFIA is a popular first-level screening tool. It has been wellknown for the attractive property of rapid and easy operation, economical and sensitive analysis (Anfossi, Baggiani, Giovannoli, & Arco, & Giraudi, 2013; Dzantiev, Byzova, Urusov, & Zherdev, 2014). However,
Corresponding author. E-mail address: [email protected] (B. Wang).
https://doi.org/10.1016/j.foodchem.2019.126055 Received 26 June 2019; Received in revised form 19 November 2019; Accepted 11 December 2019 Available online 14 December 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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the LFIA also have shortcomings, including lack of automated documentation, subjective interpretation of results, as well as inaccurate quantification (Posthuma-Trumpie, Jakob, & Aart, 2009). To overcome the abovementioned limitations, both handheld and portable LFIA readers were developed in recent years, which fulfilled the features of on-site quantitative analysis. For commercial LFIA, the colloidal gold nanobead (CGN) is the most commonly used label. It is inexpensive to prepare and can be visualized for quantitation (Mak, Beni, & Turner, 2016). On the contrary, the time-resolved fluorescent nanobead (TRFN), lanthanide Eu(III) labeled, applied in the LFIA has been increasingly reported in recent years. It possesses very large Stoke’s shifts and much longer decay times than traditional fluorescent reagents, and distinguished the useful signal from the nonspecific fluorescence signal. Therefore, it has been considered as a new quantitative screening tool for the target analyst (Goryacheva, Lenain, & De Saeger, 2013; Qi, Qu, Chen, Liu, & Li, 2017). A large number of research papers had appeared in recent years describing the development of LFIA with TRFN probe, of which the sensitivity improved greatly in comparison with the CGNLFIA (Fu, Chu, Zhao, Li, & Deng, 2017; Liu et al., 2012; Wang et al., 2015; Zhang, Li, Zhang, & Zhang, 2011; Zhang et al., 2009). In the present study, based on an ultrasensitive mAb to IMI, applying CGN and TRFN tracers for optical signal output, we developed two LFIAs for quantitation of IMI residues. The sensitivity, specificity and reproducibility of the two LFIAs were assessed and compared under an optimum condition. The suitability of LFIAs for on-site quantitation of IMI in vegetables was evaluated with a simple extract method and the portable optical readers.
2.3. Preparation of antibody probes
2. Materials and methods
The NC membrane was attached to the center of the PVC plate. Goat anti-mouse IgG (Control line, C line) and IMI-BSA (Test line, T line) were immobilized on the NC membrane at 1 μL/cm and separated by a distance of 0.5 cm. The conjugate pad was saturated with the probemAb conjugate and the sample pad was pasted on the downstream of the NC membrane sequentially. The absorption pad was pasted on the upstream. Each pad was pasted with a 0.2 cm overlap with the adjacent pad. The individual strips were placed in plastic cases, sealed in aluminum foil, and stored at 4 °C.
2.3.1. Preparation of CGN-mAb probe CGN particle (30 nm) was adjusted to pH 7.8 with potassium carbonate solution. The mAb aqueous solution was then added to the CGN solution (6 µg/mL) and stirred for 10 min. The coated CGN-mAb suspension was stabilized by adding 10% (w/v) BSA and stirred for a further 10 min, followed by centrifugation at 10,000 rpm for 10 min. The supernatant was discarded, and the CGN-mAb conjugate was resolved with Na2HPO4-KH2PO4 buffer (pH 7.4, 0.01 M) to obtain a 1% volume of the original CGN solution. 2.3.2. Preparation of TRFN-mAb probe To activate the nanobeads, 1 mL of Eu(III)-nanobeads was added to 9 mL of ddH2O, followed by adding 0.4 mL of fresh 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide solution (50 mg/mL, dissolved by ethanol respectively) to the mixture. The mixture was shaken for 30 min at room temperature and then centrifuged at 10,000 rpm for 20 min. The precipitate was suspended in 10 mL of ddH2O by ultrasonication. Next, 1 mL of mAb (1 mg/mL) was added dropwise to the activated nanobead for conjugation. The solution was shaken for 1 h at room temperature and then blocked with 10% BSA for another 1 h. The mixture was centrifuged at 10,000 rpm for 20 min and the precipitate was re-suspended in 5 mL of boric acid buffer (0.05 M, pH 8.5) containing 0.5% Tween-20 and 0.2% BSA. 2.4. Fabrication of strip
2.1. Materials and equipment The IMI standard and its analogues, bovine serum albumin (BSA), goat anti-mouse IgG, and PEG 2000 were purchased from Sigma (Shanghai, China). CGN (0.01% , w/v), nitrocellulose (NC) membranes (Sartorius CN140), sample pads, absorbent pads, polyvinyl chloride (PVC) plate and plastic cases were purchased from Jieyi Biotech Co., Ltd. (Shanghai, China). TRFN (1%, w/v) and a Microdetection® fluorescent scanner were purchased from Nanjing Microdetection Biotech Co., Ltd. (Nanjing, China). The colloidal gold scanner was purchased from Shanghai Jiening Biotech Co., Ltd (Shanghai, China). All other reagents were purchased from Beijing Chemical Reagents Co. (Beijing, China).
2.5. Optimization of LFIA Checkerboard titrations were conducted with various concentrations of IMI-BSA and probe-mAb to achieve the best sensitivity and detectability of signals. The kinetic analysis of the LFIAs was applied to obtain a best reaction time for repeatable quantitative analysis. The solvent effect with the organic solvents was optimized for the loading buffer.
2.2. Development of monoclonal antibody against IMI The hapten II of IMI from our previous work (Li & Li, 2000) was conjugated with BSA (IMI-BSA) to obtain an immunogen as previously reported (Li & Li, 2000; Lee, Ahn, Park, Kang, & Hammock, 2001). The monoclonal antibody (mAb) against IMI was produced using the hybridoma antibody technology (Guo et al., 2016). Briefly, a Bal b/c mouse was injected with immunogen for four times, and then the spleen cell was applied for fusion with murine SP 2/0 myeloma cells via PEG 2000. With an ELISA screening procedure, the monoclonal hybridoma cell line was selected to prepare ascites, and the anti-IMI mAb was further purified by the ammonium sulfate technique and stored at –40 °C before use. The animal experimental was performed strictly according to the standards described in the Guide for the Care and Use of Laboratory Animals (National Research Council Commission on Life Sciences, 1996 edition). All the animal treatment procedures were approved by the Animal Care Committee of China Agricultural University.
2.5.1. Optimization the concentration of probe-mAb on the conjugation pad The CGN-mAb conjugate solution was diluted 20-, 30-, 40-, and 50fold with PBS and then sprayed onto the conjugate pad, respectively. For the TRFN-mAb conjugate solution, the dilutions of 20-, 30-, 40-, and 50-fold with boric acid buffer (0.05 M, pH 8.5) were prepared, and then sprayed onto the conjugate pad. 2.5.2. Optimization the concentration of IMI-BSA on the T line The IMI-BSA at concentrations of 0.6, 0.8 and 1.0 mg/mL were immobilized on the T line. The C line was immobilized with 0.5 mg/mL of goat anti-mouse IgG. The NC membrane was then combined with the conjugate pad with serial contents of probe-mAb to construct strips. A volume of 100 µL PBS containing 0 ng/mL and 1 ng/mL IMI standard was pipetted into the sample well of the LFIAs. The optical signals of the T line and C line of the LFIAs were read and recorded by a stripe reader. 2.5.3. Optimization of the reaction time An aliquot of 100 µL of PBS containing 0 ng/mL and 1 ng/mL IMI 2
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standard was pipetted into the sample well of the LFIAs. After 2 min of incubation, the signal intensity of the LFIAs was read by the strip reader every 2 min. The signal intensity ratio of T line to C line against time was plotted to establish the kinetic curves. Each measurement was performed in triplicate.
filtered through 0.22 μm filter prior to analysis. 2.7.3. LC-MS/MS analysis A Shim-pack XR-ODS III column (150 mm × 2.0 mm, i.d., 2.2 μm; Shimadzu Corp., Japan) was applied in the column oven kept at 40 °C. The injection volume was 1 μL. The mobile phase with a flow rate of 0.4 mL/min consisted of eluent A (methanol) and eluent B (1 mmol/L aqueous ammonium acetate). The mobile phase was started with 10% A and 90% B, The proportion of A linearly increased to 50% over 4 min, and then to 75% over 16 min. The proportion of A was increased to 95% over 2 min and held for 5 min, then immediately adjusted to its initial composition and held for 8 min. The retention time of IMI was 5.27 min. For MS analysis, the electrospray ionization source was operated in both positive and negative mode (4,000 V). High-purity N2 served both as sheath and auxiliary gas. Argon (2.7 × 105 Pa) was used as the collision-induced dissociation gas. Detection was carried out in the MRM mode. The mass spectroscopy transitions and fragmentation conditions selected for IMI are m/z 256.1 → 175.1 at −16 eV.
2.5.4. Optimization of the loading buffer Methanol and acetonitrile were often used to extract IMI from matrices. Effects of methanol and acetonitrile were studied using PBS containing 0, 5, and 10% of the organic solvents to dilute the IMI standard. These solutions were pipetted into the sample well of the LFIAs. The optical signals of the T line and C line were recorded. Each measurement was performed in triplicate. 2.6. Evaluation of the LFIA 2.6.1. Standard curves A stock solution of IMI was diluted with PBS to a series of concentrations for standard solutions. Each solution was added into the sample well of LFIA. After incubation, the optical intensity ratio of the T line to the C line was read for each concentration as B. The standard curve was obtained by plotting the ratio of the B value to the B0 value (T/C of the 0 ng/mL) versus the logarithm of IMI concentration, and they were fitted into a four-parameter logarithmic equation. The IC50 value was taken as the assay sensitivity. The linear working range was represented by IC20-IC80 (Van Emon, 2007). The IC10 was used to calculate the limit of detection (LOD) as previously reported (Brady, 1995). Each measurement was performed in triplicate. The standard curve generation and data treatment were performed with OriginPro 8.1 (OriginLab).
3. Results and discussion 3.1. Development of monoclonal antibody against IMI With the hybridoma antibody technology, we obtained the anti-IMI mAbs. The best clone, designated as 4D9, was used to produce ascites. The ascites titer was 8 × 105 and further purified by the ammonium sulfate technique. Using the mAb 4D9, an inhibition curve obtained by ELISA was displayed as short dot line in Fig. 3(A). The IC50 was calculated as 0.07 ng/mL, while the dynamic range was 0.024–0.20 ng/ mL.
2.6.2. Recovery A standard recovery towards IMI was recorded to evaluate the accuracy of LFIA in vegetable samples. After determined as IMI-free samples via an LC-MS/MS method, IMI-free vegetable samples were spiked with IMI standard solution to achieve concentration gradient series. Ten microliter of these serial IMI dilutions were spiked into IMIfree samples, and then extracted and determinate by the LFIAs. The recovery was calculated as follows: Recovery (%) = (Measured amount/Spiked amount) × 100%. Each assay repeated three times.
3.2. Optimization of LFIA Checkerboard titrations were applied to optimize the concentration of IMI-BSA and probe-mAb. The results (Fig. 1) were expressed as the dilution fold of the labeled nanobeads against the T/C value. The black line and gray line displayed the inhibition trend of 0 ng/mL and 1 ng/ mL of IMI, respectively. For the CGN-LFIA (Fig. 1A), the pair, 0.8 mg/ mL of IMI-BSA and 30 × of labeled CGN-mAb, was chosen for subsequent study. Under this condition, the inhibition ratio could achieve the highest value, and it could give a minimum T/C of 1 ng/mL while maximum T/C of 0 ng/mL. For the same seasons, we chose the pair, 0.6 mg/mL of IMI-BSA and 40 × of labeled TRFN-mAb for the TRFNLFIA (Fig. 1B). To compare the two LFIAs, the CGN-LFIA was applied with approximately 2 ng of CGN for one strip, and it was approximately 0.4 ng of TRFN for the other one. While the optical signals were detected in an available range, the IMI-BSA usage of TRFN-LFIA was also less than that of CGN-LFIA, which was consistent with the previous reports (Hu et al., 2017; Pyo, 2007). The optimization of reaction time was performed by recording the optical signal every 2 min after pipetting the standard solution into the sample well. As Fig. 2 shows, both the signal intensity of CGN- and TRFN-LFIA had enhanced over time (inset graph), On the contrary, the T/C value decreased. The signal variation of 0 ng/mL was greater than that of 1 ng/mL. Both features of CGN-LFIA raised and fell more greatly than the TRFN-LFIA. Ten min after addition of the standard solution into the sample well, the optical intensity and T/C value of the TRFNLFIA were relatively constant, which enabled a long time for scanner reading. The continuous decrease of the T/C value of the CGN-LFIA showed that 10–15 min after sample addition could allow the scanner reading. The organic solvents in the immunoreaction medium often influence the analyte-antibody interaction, causing considerable interference in the assay. Thus, the effect of methanol and acetonitrile on the LFIA was evaluated in the present research. The two organic solvents in PBS at concentrations of 5% and 10% were prepared to dilute IMI for standard
2.6.3. Specificity The specificity of the LFIA for IMI was evaluated by cross-reactivity (CR). Stock solutions of clothianidin, acetamiprid, thiamethoxam, dinotefuran, and 6-chloronicotinic acid were separately prepared with PBS and pipetted to the sample well of the LFIAs. The CR was calculated according to the formula: CR (%) = (IC50 of IMI) / (IC50 of analyte) × 100. Each sample was analyzed in triplicate. 2.7. Validation of LFIA Chinese leek samples were collected from local markets, analyzed by the LFIAs and further confirmed using LC-MS/MS. Each sample was analyzed in triplicate. 2.7.1. Sample preparation for LFIA The samples were finely ground and 1 g sample was extracted in 5 mL PBS. After shaking for 10 min at room temperature, the extract was stood for 5 min, and then the supernatant was diluted with PBS before sensing. 2.7.2. Sample preparation for LC-MS/MS The sample preparation for LC-MS/MS was performed as previously described (Li, 2016). Ten grams of sample were extracted in 20 mL of acetonitrile by shaking for 30 min. Five grams of NaCl were added, followed by centrifugation at 5,000 rpm for 3 min. The supernatant was 3
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Fig. 1. Optimization of the concentration of IMI-BSA, probe-mAb (A and B) and the loading buffer (C and D).
curve (Fig. 1C and D). The two organic solvents caused mass variations of the standard curve. To avoid the effect of organic solvents, the solvents should be removed prior to the test.
3.3. Evaluation of the LFIA 3.3.1. Standard curves The standard curves of the two optimized LFIAs were constructed by plotting B/B0 against the logarithm of IMI concentration. Fig. 3 shows a
Fig. 2. Optimization of reaction time for colloidal gold lateral flow immunoassay (CGN-LFIA) and time-resolved fluorescent nanobead traced lateral flow immunoassay (TRFN-LFIA). 4
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interaction, and it is often easy and routine to perform. The TRFN and mAb were conjugated via chemical bonds. Although it is well known that chemical bond is stronger than the electrostatic interactions, the reaction for TRFN was tedious and time-consuming. Moreover, once the reaction condition was inappropriate, the precipitation appeared frequently during the reaction and was difficult to disperse. An ELISA assay was also compared with the two LFIAs. The CGNLFIA could give a full coverage of ELISA assay range. An integration of an optical scanner with the CGN-LFIA could fully replace the ELISA for one-step quantitation of IMI. 3.3.2. Recovery To test the reliability of the developed assay in practical application, Chinese leek, sweet potato and potato were fortified with IMI at concentrations of 0–40 ng/g. The average recoveries of IMI detected with CGN-LFIA were 72.3–106.3%, 69.1–111.3% and 58.6–121.4% for Chinese leek, sweet potato and potato, respectively (Table 1). The corresponding average recoveries measured with TRFN-LFIA were 78.8–108.6%, 58.1–88.7%, and 72.7–91.1%. The two major facts, extraction buffer containing no organic reagent and short extraction time, allowed the LFIA more suitable for on-site test. In addition, the spike tests showed that the IMI content below 0.5 ng/g could not be detected in vegetable samples with the LFIAs, which was far less than the detection potential. The reason might come from the simple sample preparation, which no presence of organic reagents in the extraction buffer and short extraction time allowed the LFIA more suitable for on-site tests. To improve the LOD of IMI in real samples, extract cleanup should be considered. 3.3.3. Specificity To evaluate the specificity of the LFIAs, IMI analogues (clothianidin, acetamiprid, thiamethoxam, and dinotefuran) and major metabolite 6chloronicotinic acid were used in the CR study (Table 2). The CR of clothianidin and acetamiprid were 3.6% and 1.1% via CGN-LFIA, respectively, and which were calculated as 4.1% and 1.1% using the TRFN-LFIA, respectively. There was no inhibition even at the concentration of 1000 ng/mL of thiamethoxam, dinotefuran, and 6-chloronicotinic acid. Thiamethoxam, dinotefuran, and 6-chloronicotinic acid could not disturb the assay, while clothianidin and acetamiprid only at high concentrations affected the test results.
Fig. 3. (A) Standard curves of imidacloprid detected by colloidal gold lateral flow immunoassay (CGN-LFIA), time-resolved fluorescent nanobead traced lateral flow immunoassay (TRFN-LFIA) and ELISA. (B) CGN-LFIA and (C) TRFN-LFIA strips showing color changes corresponding to concentrations of imidacloprid in PBS.
3.4. Validation of LFIA To further confirm the accuracy of the developed LFIAs, Chinese leek samples were collected and analyzed by the CGN-LFIA, TRFN-LFIA and LC-MS/MS. Table 3 shows that the LC-MS/MS data correlated well with those from LFIAs (R2, 0.91). Bland-Altman bias plots are provided in Supplemental File for comparison between the LFIA and LC-MS/MS. The IMI residue content in Chinese leek samples detected with LFIAs was between 0.7 and 50 ng/g. Samples with IMI content > 2 ng/g were verified by LC-MS/MS. The content of the sample No. 1 detected by LFIAs was 0.7 ng/g and 0.8 ng/g, which were not detected with the LCMS/MS method. The reason for this may be the sample content lower than the LOD of LC-MS/MS, or the detection result of LFIA was false positive. The commercial potato, sweet potato and most Chinese leek samples were negative when LFIAs and LC-MS/MS were used. According to the China National Food Safety Standard-Maximum Residue Limits (MRL) for Pesticides in Food (GB 2763-2016), the MRLs of IMI in vegetable range from 0.2 to 5 mg/kg. The European Commission set the MRL of 0.05 mg/kg for IMI in leek. MRLs of 0.02–5 ppm for IMI was established for the sum of the IMI residue on agricultural products by the Japan Food Chemical Research Foundation, and MRL of 0.4 ppm for IMI residue in potato vegetable was currently in effective in the United States (no MRL for leek). After unit conversion (0.7–50 ng/g equivalent to 0.7 × 10−3-0.05 mg/kg and ppm), obviously, the content of the positive leek samples presented
typical inhibition curve of the CGN-LFIA (black curve). The regression equation was Y = −0.07 + 1.07/[1+(X/0.15)0.889] with a reliable correlation coefficient R2 = 0.998. The IC50, LOD and the dynamic range were 0.13 ng/mL, 0.01 ng/mL, and between 0.028 ng/mL and 0.50 ng/mL. The inhibition curve of the TRFN-LFIA was showed as the gray curve, with an IC50 of 0.14 ng/mL, a LOD of 0.02 ng/mL, and a dynamic range of 0.039–0.54 ng/mL. The regression equation was Y = 0.05 + 0.93/[1+(X/0.13)1.165] and the correlation coefficient R2 was 0.992. Although the ELISA possessed the smallest value of IC50 among the three assays, the detection range of CGN-LFIA was larger than the other two methods. The CGN-LFIA could fully replace the ELISA when it is used to detect IMI in actual samples. Contrary to earlier reports that TRFN was capable of delivering sensitivity in a greater range (4–5 fold) than CGN (Hu et al., 2017; Pyo, 2007), there was no significant difference in the IC50 and detection range between the two LFIAs in the present study. The TRFN tracer seemed no remarkable superiority. The CGN-LFIA could achieve the same sensitivity as the TRFN-LFIA. Moreover, the LODs of the reported TRFN-LFIA assay were rare to find lower than 10 ng/L for small molecules. The conjugation of CGN and mAb relies on electrostatic 5
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Table 1 Average recoveries of imidacloprid in vegetable samples measured by colloidal gold lateral flow immunoassay (CGN-LFIA) and time-resolved fluorescent nanobead traced lateral flow immunoassay (TRFN-LFIA). Sample
Chinese leek
Sweet potato
Potato
Fortified (ng/g)
0 0.5 1 10 20 40 0 0.5 1 10 20 40 0 0.5 1 10 20 40
CGN-LFIA
TRFN-LFIA
Detected (ng/g)
Recovery (%)
SD
Detected (ng/g)
Recovery (%)
SD
ND 0.53 0.72 9.15 15.96 37.74 ND 0.56 0.69 7.13 18.14 38.81 ND 0.61 0.59 7.70 15.03 28.45
ND 106.3 72.3 90.6 79.8 94.3 ND 111.3 69.1 71.3 90.7 97.0 ND 121.4 58.6 77.0 75.1 71.1
ND 12.5 15.2 2.3 0.7 6.1 ND 28.5 1.6 0.4 4.1 7.6 ND 14.2 4.1 0.4 0.6 1.6
ND 0.46 0.82 10.86 19.13 31.52 ND 0.29 0.67 8.87 14.73 33.08 ND 0.38 0.73 8.54 14.58 36.45
ND 91.8 82.2 108.6 95.6 78.8 ND 58.1 67.3 88.7 73.6 82.7 ND 76.5 72.7 85.4 72.9 91.1
ND 3.0 9.6 2.4 3.4 1.4 ND 2.9 6.2 0.4 0.0 2.2 ND 1.4 2.3 1.7 2.3 4.1
in this paper were far less than the MRLs mentioned above. The extract of sample containing 0.02 mg/kg of IMI could be diluted 200 fold to meet the threshold value of standard curves, which ensured the elimination of matrix interference.
in real samples with the LFIAs. Further purification of sample extracts may improve the LOD. The good agreement was found between the LFIAs and LC-MS/MS for the analysis of IMI in commercial Chinese leek. The LFIAs can be used as a routine method for monitoring IMI residues in vegetables.
4. Conclusions
Author Contributions
In the present study, we developed two types of LFIAs with an ultrasensitive mAb. The assay sensitivities for IMI were greater than most reported studies and as sensitive as the ELISA. The IC50 and LOD were 0.13 and 0.01 ng/mL for CGN-LFIA, and 0.14 and 0.02 ng/mL for TRFN-LFIA, respectively. The TRFN-LFIA and CGN-LFIA had a similar LOD. The two assays had the average recovery of 58–121% for spiked IMI in vegetables. The IMI content below 0.5 ng/g could not be detected
Guiyu Tan developed the monoclonal antibody against imidacloprid, established the two lateral flow immunoassays. Yajie Zhao collected and prepared the sample and preformed the LC-MS/MS analysis. Mian Wang prepared the conjugate of hapten-carrier protein and probeantibody. Xiaojiao Chen evaluated the lateral flow immunoassays. Baomin Wang planned experiments, analysed data and supervised the
Table 2 Specificity of colloidal gold lateral flow immunoassay (CGN-LFIA) and time-resolved fluorescent nanobead traced lateral flow immunoassay (TRFN-LFIA) to imidacloprid and its analogs. Analyte
Structure
CGN-LFIA
TRFN-LFIA
IC50 (ng/mL)
Cross reactivity (%)
IC50 (ng/mL)
Cross reactivity (%)
Imidacloprid
0.13
100
0.14
100
Clothianidin
3.62
3.6
3.43
4.1
Acetamiprid
12.22
1.1
12.79
1.1
Thiamethoxam
> 5000
< 0.01
> 5000
< 0.01
Dinotefuran
> 5000
< 0.01
> 5000
< 0.01
6-Chloronicotinic acid
> 5000
< 0.01
> 5000
< 0.01
6
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Table 3 Concentrations of imidacloprid in commercial Chinese leek determined with colloidal gold lateral flow immunoassay (CGN-LFIA), time-resolved fluorescent nanobead traced lateral flow immunoassay (TRFN-LFIA) and LC-MS/MS. Sample No.
CGN-LFIA (ng/g)
TRFN-LFIA (ng/g)
LC-MS/MS (ng/g)
1 2 3 4 5 6 7 8 9 10
0.8 ± 0.2 ND ND ND 37.6 ± 2.4 56.6 ± 2.9 ND ND 8.6 ± 0.6 15.8 ± 0.0
0.7 ± 0.2 ND ND ND 27.5 ± 3.2 41.3 ± 2.6 ND ND 9.2 ± 1.2 13.4 ± 0.5
ND ND ND ND 28.6 ± 1.1 47.7 ± 0.8 ND ND 7.8 ± 0.4 14.6 ± 0.8
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project. Qing X. Li prepared the hapten. Guiyu Tan, Baomin Wang and Qing X. Li wrote the manuscript with contributions from all other authors. Funding This work was supported by the National Key Research and Development Program of China [grant number 2018YFC1602900] and the Guangxi Science and Technology Major Project [grant number AA17204043]. Declaration of Competing Interest 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. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.126055. References Anfossi, L., Baggiani, C., Giovannoli, C., D‘Arco, G., & Giraudi, G. (2013). Lateral-flow immunoassays for mycotoxins and phycotoxins: A review. Analytical & Bioanalytical Chemistry, 405(2–3), 467–480. Bai, D., Lummis, S. C. R., Leicht, W., Breer, H., & Sattelle, D. B. (2010). Actions of imidacloprid and a related nitromethylene on cholinergic receptors of an identified insect motor neurone. Pest Management Science, 33(2), 197–204. Brady, J. F. (1995). Interpretation of immunoassay data. Acs Symposium, 266–287. Buckingham, S., Lapied, B., Corronc, H., & Sattelle, F. (1997). Imidacloprid actions on insect neuronal acetylcholine receptors. Journal of Experimental Biology, 200(Pt 21), 2685–2692. Dzantiev, B. B., Byzova, N. A., Urusov, A. E., & Zherdev, A. V. (2014). Immunochromatographic methods in food analysis. Trends in Analytical Chemistry, 55(55), 81–93. Fang, Q., Wang, L., Cheng, Q., Cai, J., Wang, Y., Yang, M., ... Liu, F. (2015). A bare-eye based one-step signal amplified semiquantitative immunochromatographic assay for the detection of imidacloprid in Chinese cabbage samples. Analytical Chimica Acta, 881, 82–89. Fu, X., Chu, Y., Zhao, K., Li, J., & Deng, A. (2017). Ultrasensitive detection of the βadrenergic agonist brombuterol by a SERS-based lateral flow immunochromatographic assay using flower-like gold-silver core-shell nanoparticles. Microchimica Acta, 184(6), 1711–1719. Goryacheva, I. Y., Lenain, P., & De Saeger, S. (2013). Nanosized labels for rapid immunotests. Trends in Analytical Chemistry, 46, 30–43.
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