A rapid immunomagnetic-bead-based immunoassay for triazophos analysis

Abstract

An immunomagnetic-bead-based enzyme-linked immunosorbent assay (IMB-ELISA) was developed for detection of pesticides by using carboxyl functionalized magnetic Fe₃O₄ nanoparticles (CMNPs). The CMNPs were prepared by co-precipitation of Fe²⁺/Fe³⁺ with oleic acid as a surfactant and subsequent oxidation of C=C into COOH by KMnO₄ solution in situ. Then, anti-pesticide (triazophos) monoclonal antibodies were directly bonded onto the magnetic nanoparticles, which significantly increased the sensitivity compared with classic ELISA. The detection limit was 0.10 ng mL⁻¹. Addition-recovery and high-precision experiments were performed on blank samples that were determined to be without triazophos. The average recovery rate for three types of samples (with each spiking concentration measured 5 times in parallel) ranged from 83.1% to 115.9%, with a relative standard deviation (RSD) of less than 10%, which meets the requirement of pesticide residue analysis. The application results were in accordance with the gas chromatography-mass spectrometry (GC-MS) method, suggesting that IMB-ELISA is rapid and reliable for pesticide detection.

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1. Introduction

Food safety is of critical concern for people all around the world. One of the key factors causing food security issues is pesticide residues,^1–4 which are of great significance. Many countries have drafted strict pesticide residue limit standards, which ensure proper and safe use of pesticides and serve as technical barriers to trade to benefit their own countries. Triazophos(O,O-diethyl-O-(1-phenyl-l,2,4-triazole-3-yl)phosphorothioate) is a type of broad-spectrum insecticide, which is highly effective for pest control on main agricultural products such as grains, fruits, and vegetables, cotton, oil, and tea.^5–8 Triazophos inhibits acetylcholinesterase and influences central nervous system (CNS) functions. It is highly toxic to fish, bees, and silkworms, and has medium toxicity to mammals.^9–12 After highly toxic organophosphorus pesticides such as methamidophos and parathion were prohibited or restricted, the use of triazophos has quickly increased in rice and other crops because it features low toxicity to humans, while maintaining high effectiveness and broad spectrum activity against important pests. However, triazophos has a long half-life period and easily remains in the environment so as to damage the environment, wildlife, and human health, which all draw human attention.¹³

Currently, the techniques and methods for detecting pesticides have been extensively studied. The analysis techniques based on instruments such as gas chromatography (GC),¹⁴ liquid chromatography (LC),¹⁵ gas chromatography-mass spectrometer (GC-MS),¹⁶ and liquid chromatography-mass spectrometer (LC-MS)¹⁷ are relatively mature and complete. However, these methods require sophisticated instrumentation, skilled analysts and time-consuming sample preparation procedures. Enzyme-linked immunosorbent assay (ELISA) has been widely applied to pesticide residue testing. Although ELISA is highly sensitive in standard detection, the lowest detection limits for some pesticides in the samples are still less than their respective maximum residue level (MRL) values defined by many countries. Immunomagnetic-bead-based immunoassay, which includes technical advantages, has been successfully applied in the field of food safety detection,^18,19 environment monitoring^20,21 and clinical diagnosis.²² Immunomagnetic beads (IMBs) have more surface areas than microtiter plates, permitting more “active molecules” to be immobilized on the surface.^23,24 IMBs can also be easily separated from reaction mixtures with a magnet and re-dispersed immediately following removal of the magnet.²⁵ IMBs allow for a nearly “in solution” reaction.²⁶ These characteristics lead to increased sensitivity and shorter reaction time.

An IMB-ELISA approach to detect pesticides generally uses noncompetitive and indirect competitive enzyme-linked immunoassay methods, which demands long time and two kinds of antibodies.^20,27 The differences between the indirect and direct formats of monoclonal-antibody-based ELISA for triazophos lie in that the direct format shows higher sensitivity and shorter time than the indirect format and is used more widely than the indirect-format ELISA.²⁸ Herein, we reported a direct competitive IMB-ELISA approach to detect the triazophos pesticides. The work demonstrated the high potential of IMB-ELISA to improve sensitivity and broad the application scope in pesticide residue.

2. Experimental

2.1 Chemicals and reagents

The vibrating sample magnetometer (VSM) EV7 was purchased from ADE (US). Spectrum One Fourier transform infrared spectrometer was obtained from PerkinElmer. Polycrystalline X-ray diffractometer was from Bruker (D8 ADVANCE, Germany). TECHAI-12 transmission electron microscope (TEM) was from Philips, The Netherlands. Sunrise microplate reader was purchased from Tecan, and the magnetic separation rack was purchased from the LanGang Biotech company. 96-well plates were purchased from Corning Costar (Acton, MA). Agilent 7890A/GS-MS instrument was from Agilent, America.

The triazophos standard, 2-(N-morpholino)ethanesulfonic acid (MES), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), N,N-dimethylformamide (DMF), N,N′-dicyclohexyl carbodiimide (DCC), albumin from bovine serum (BSA) and horseradish peroxidase (HRP) were purchased from Sigma-Aldrich (St. Louis MO, USA). Primary secondary amine (PSA) and C₁₈ solid-phase extraction packing materials were purchased from Bonna-Agela Technologies (Tianjin, China). Acetonitrile and methanol of the HPLC grade were acquired from Thermo Fisher Scientific (MA, USA). All other chemicals and organic solvents, including Tris, hydrochloric acid, Tween 20, and peroxide, were of analytical grade or better and were purchased from Beijing chemical industry group Co., Ltd (Beijing, China).

Blank matrices of the apple, orange, litchi, cabbage, zucchini and rice were purchased from local supermarkets, which were of high quality and pollution-free, as confirmed by GC-MS.

2.2 Sample preparation

Blank samples (apple, orange, litchi, cabbage, zucchini, and rice) were mashed and then stored in a refrigerator at −20 °C. For recovery studies, the pesticide-free apple, orange, litchi, cabbage, zucchini, and rice were spiked with triazophos. The solutions of the pesticide in methanol used to fortify the samples were prepared. The fortifying solution was added to 10 g of finely chopped apple, orange, litchi, cabbage, zucchini, rice. After the samples were set aside for 24 h, 3 mL of water was added into a 50 mL plastic centrifuge tube. Acetonitrile extraction solution was added to obtain a constant volume of 10 mL for 10 min with vigorous shake. 4 g of anhydrous MgSO₄ plus 1 g of NaCl was added, vortexed immediately and centrifuged at 10 000 rpm for 5 min at 4 °C. 2 mL of acetonitrile extraction solution was accurately transferred into a micro-centrifuge. Subsequently, 50 mg of PSA and 50 mg of C₁₈ dispersive solid-phase extraction purifier were added, and then it was centrifuged at 10 000 rpm for 5 min under 4 °C after high-speed vortex for 0.5 min. 1 mL of the supernatant was blown to dryness at 30 °C under nitrogen. Finally, the residue was dissolved in 5% methanol–PBS. IMB-ELISA was conducted to analyze the extract, and the recovery was determined using the standard curve obtained from the standards in methanol–PBS.

2.3 Optimization of the IMB-ELISA

Dilutions of immunomagnetic beads, concentrations of the monoclonal antibody and hapten-HRP were optimized by chessboard design with an OD value of about 1.0 in IMB-ELISA. The OD value at 450 nm (OD₄₅₀) for different dilutions of immunomagnetic beads (1 : 5, 1 : 10, 1 : 20, 1 : 30, 1 : 40, and 1 : 80) and different concentrations of hapten-HRP (6, 3, 1.5, 0.750, 0.375, 0.188, 0.094, and 0.047 μg mL⁻¹) were tested, except that no triazophos was added.

2.4 Process of immunoassay

All ELISA tests were carried out in the same manner. Blank wells (zero antibody or no coating conjugate) and controls were incorporated for measurement of the maximum signal (zero analyte) and non-specific binding. The general procedures are described as follows.²⁹

IMB-ELISA is performed as follows: 100 μL of IMB probes were diluted 1 : 20 with the binding buffer in the Eppendorf tubes. Triazophos standards (0.078, 0.16, 0.31, 0.63, 1.25, 2.5, 5.0, and 10 ng mL⁻¹) in 10% (v/v) methanol–PBS (50 μL) or samples (50 μL) and 50 μL enzyme tracer hapten-HRP solutions were added to the EP tubes. After 30 min incubation at 37 °C, the conjugates were precipitated on the bottom of the EP tubes and the supernatant was discarded by magnetic force. The IMBs were then re-dispersed with 200 μL of washing buffer and washed three times. After the IMBs were transferred to a new Eppendorf tube, 100 μL of substrate solution was added (TMB liquid substrate system). After incubation for 10–15 min at room temperature, color development was stopped with 50 μL of H₂SO₄ (2 mol L⁻¹). The supernatant was collected with a magnetic separator, transported to a 96-well plate and measured at 450 nm.

Classic ELISA is performed as follows: the plate wells were coated with 100 μL of triazophos monoclonal antibodies (McAbs) solution (1 : 1000 in PBS) at 37 °C for 2 h. After the plate was washed three times with PBST (PBS containing 0.05% Tween 20, pH 7.4), free binding sites of the wells were blocked with 300 μL per well of 2% BSA for 30 min at 37 °C. Triazophos standards (0.078, 0.16, 0.31, 0.63, 1.25, 2.5, 5.0, and 10 ng mL⁻¹) in 10% (v/v) methanol–PBS (50 μL) or samples (50 μL) and 50 μL of enzyme tracer hapten-HRP solutions were added to the wells. After 60 min incubation at 37 °C, the plates were washed, 100 μL of substrate solution was added (TMB liquid substrate system). After incubation for 10–15 min at room temperature, color development was stopped with 50 μL of H₂SO₄ (2 mol L⁻¹) and the absorbance was measured at 450 nm.³⁰

Standard curves were plotted as the inhibition against the analyte concentration.

2.5 Chromatography-mass spectrometry conditions

Gas chromatography-tandem mass spectrometry (GC-MS) was performed in the following conditions: column: DB-5MS (30 m × 250 μm × 0.25 μm) silica capillary column; oven temperature procedure: 60 °C maintained for 4 min, heated by 30 °C min⁻¹ to 180 °C, and then heated to 250 °C at the speed of 10 °C min⁻¹ and kept for 4 min; carrier gas: helium, purity ≥ 99.999%, constant voltage mode, and 7.136 psi pressure; injection temperature: 220 °C; injection volume: 10 μL; injection mode: splitless injection, and the by-pass valve and septum purge valve opened 1.0 min later; electron impact ionization (EI): 70 eV; ion source temperature: 200 °C; GC-MS interface temperature: 250 °C.

3. Results and discussion

3.1 Morphology and particle size analysis

The particle size distribution and morphology of magnetic Fe₃O₄ nanoparticles were analyzed using a scanning electron microscope, and the sample on the copper disposed by metal spraying was observed. Fe₃O₄ magnetic nanoparticles diluted with deionized water were treated by ultrasonic technique, and a drop was added to a special copper mesh coated with the carbon support film and then analyzed by a transmission electron microscope after drying. After the two pictures in Fig. 1 were compared, we can conclude that there is no significant change in the particle size upon modification, and the dispersibility of the modified particles is better.

Fig. 1 TEM images of Fe₃O₄ nanoparticles (right) and carboxyl-functionalized Fe₃O₄ nanoparticles (left).

3.2 Optimization of direct competitive IMB-ELISA

The data of the checkerboard method were shown in Table 1. The dose of immunomagnetic beads had a significant effect on the assay performance. Based on the experiment, the optimum dilution of immunomagnetic beads was selected as 1 : 20, and the concentration of enzyme-labeled haptens was chosen as 0.750 μg mL⁻¹.

Table 1 Chessboard titration of IMB and hapten-HRP

Concentration of hapten-HRP (μg mL^−1)	Dilution ratio of IMB (μg mL^−1)
	1 : 5	1 : 10	1 : 20	1 : 40	1 : 80	1 : 100	Negative control
3.000	2.978	2.778	2.352	1.411	0.420	0.146	0.035
1.500	2.030	1.990	1.576	0.887	0.274	0.134	0.041
0.750	1.641	1.241	1.094	0.511	0.184	0.097	0.023
0.375	0.938	0.638	0.561	0.294	0.124	0.097	0.035
0.188	0.860	0.360	0.233	0.174	0.113	0.083	0.037
0.094	0.404	0.204	0.163	0.138	0.093	0.090	0.026
0.047	0.226	0.126	0.119	0.106	0.078	0.075	0.020

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3.3 Protocol of the assay

The basic principle of IMB-ELISA was illustrated in Fig. 2. The hapten-HRP conjugates were used as detection probes, the conjugates of McAbs of triazophos and carboxylated magnetic nanoparticles were used as capture probes, and the triazophos pesticides to be detected became the competitors of the ELISA hapten. The HRP can catalyze the coloration of the substrate TMB so as to detect triazophos pesticides. The absorbance and color are positively correlated to each other.

Fig. 2 Schematic illustration of competitive reaction (A) and IMB-based immunoassay procedure (B).

3.4 Standard curve

The standard curve of IMB-ELISA was drawn based on the optimized conditions in Table 1. The detection limit was calculated by IC₁₀ (10% inhibition in the maximal OD value). The concentrations of the triazophos standard samples were set as 0.078, 0.16, 0.31, 0.63, 1.25, 2.5, 5.0, and 10 ng mL⁻¹. According to the inhibition rate formula, the average inhibition rate for each concentration was calculated. Then, a standard curve was established for statistical analysis. Fig. 3 shows the standard curve of the classic ELISA competitive reaction for triazophos. Fig. 4 shows the standard curve of the IMB-ELISA competitive reaction for triazophos. With the analyte concentrations and corresponding average inhibition rates, the regression equation of IMB-ELISA for the quantification of triazophos was y = 0.1701 ln(x) + 0.485 (R² = 0.9917), and the detection limit and IC₅₀ were 0.10 ng mL⁻¹ and 1.09 ng mL⁻¹, respectively. The regression equation of classic ELISA was y = 0.1194 ln(x) + 0.279 (R² = 0.9756), and the detection limit and IC₅₀ were 0.22 ng mL⁻¹ and 6.36 ng mL⁻¹, respectively. Each concentration was measured 5 times in parallel. Based on the experiments and number of replicates, the data were assessed by SPSS version 13.0 software. Differences between groups were assessed by Student's t test (t test). Statistical significance was considered at p < 0.01. According to the result, IMB-ELISA has higher sensitivity than classic ELISA.

Fig. 3 Standard curve of classic ELISA competitive reaction for triazophos.

Fig. 4 Standard curve of IMB-ELISA for triazophos.

In this equation, “I%” was the inhibition rate, “OD_max” was the maximum signal (zero analyte), “OD_x” was the signal correlated with the analyte concentration, and “OD_min” was the signal of blank wells (zero antibody or no coating conjugate).

3.5 Recovery rates and the accuracy of the method

According to the MRLs of China and CAC,³¹ blank samples (apple, orange, litchi, cabbage, zucchini, and rice) were spiked with the triazophos standard. The spiking concentrations are 100 μg kg⁻¹, 200 μg kg⁻¹, and 500 μg kg⁻¹ for fruits including apples, citrus, and litchi, 50 μg kg⁻¹, 100 μg kg⁻¹, and 200 μg kg⁻¹ for vegetables including cabbages and zucchini, and 10 μg kg⁻¹, 50 μg kg⁻¹, and 100 μg kg⁻¹ for grains including rice. The results are shown in Table 2. The recovery rates with IMB-ELISA were determined as ranging from 83.1% to 115.9% for triazophos, and the RSD was less than 10%, indicating that the method met the requirement of pesticide residue analysis.

Table 2 Reproducibility and recovery of triazophos from spiked samples (n = 5)

Sample	Spiked concentration (μg kg^−1)	Recovery (%)	CV^a (% n = 5)	Sample	Spiked concentration (μg kg^−1)	Recovery (%)	CV^a (% n = 5)
a CV = coefficient of variation.
Apple	100.0	83.1	6.9	Cabbage	50.0	110.9	4.6
	200.0	94.3	4.8		100.0	97.5	5.8
	500.0	110.5	8.2		200.0	99.6	9.0
Orange	100.0	86.5	7.6	Zucchini	50.0	103.8	5.7
	200.0	109.1	8.6		100.0	96.3	4.4
	500.0	115.3	5.1		200.0	89.5	7.9
Litchi	100.0	90.2	5.6	Rice	10.0	103.3	4.9
	200.0	99.9	4.4		50.0	115.9	8.1
	500.0	103.1	6.0		100.0	96.1	6.7

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3.6 Correlation studies between IMB-LISA and GC-MS method analysis

To validate the performance of IMB-ELISA, apple and cabbage samples spiked with the triazophos standard were selected for comparison experiments. The concentrations of triazophos were simultaneously measured by the GC-MS method and developed assay, and the results were compared. Fig. 5 showed the detection results of triazophos in apple and cabbage samples by using IMB-ELISA and GC-MS. The linear regression analysis showed a good correlation between the two methods. The results indicated that IMB-ELISA is a credible immunoassay for detection of triazophos. The GC-MS detection spectrogram includes a 50 μg L⁻¹ standard solution spectrogram, an apple matrix blank spectrogram, and a recovery spectrogram, which were shown in Fig. 6.

Fig. 5 Correlation between IMB-ELISA and GC-MS for determination.

Fig. 6 Chromatograms of blank and spiked samples ((A) apple spiked with triazophos (0.1 mg kg⁻¹) (B) 50 μg L⁻¹ standard solution of triazophos (C) blank apple sample).

4. Conclusion

A very sensitive immunoassay method has been developed for triazophos determination, allowing detection of triazophos at the ng L⁻¹ level. This IMB-ELISA method was more sensitive than classic ELISA and was less time-consuming. The results of apple and cabbage samples spiked with the triazophos standard showed a good correlation between the developed assay and the GC-MS method, demonstrating that IMB-ELISA would be a reliable tool for rapid detection of triazophos. As a new strategy, IMB-ELISA is accurate, sensitive, and quick, which makes it suitable for rapid detection of triazophos in agricultural and food-safety settings.

Acknowledgements

This study was supported by the National Natural Science Foundation (31201371), Chinese Public Interest Industrial Science & Technology Project (201203094), and National Key Foundation for Exploring Scientific Instrument (2013YQ140371).

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Footnote

† Electronic supplementary information (ESI) available: Buffers and solutions, synthesis of carboxyl-functioned Fe₃O₄ magnetic nanoparticles (CMNPs), preparation of immunomagnetic beads (IMBs), preparation of hapten-HRP (horseradish peroxidase) conjugates. See DOI: 10.1039/c5ra15106f

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