Anal Bioanal Chem DOI 10.1007/s00216-017-0653-7
RESEARCH PAPER
Upconversion fluorescence immunoassay for imidaclothiz by magnetic nanoparticle separation Xiude Hua 1 & Hongjie You 1 & Peiwen Luo 1 & Zhexuan Tao 1 & He Chen 1 & Fengquan Liu 1,2 & Minghua Wang 1
Received: 10 July 2017 / Revised: 12 September 2017 / Accepted: 18 September 2017 # Springer-Verlag GmbH Germany 2017
Abstract A sensitive fluorescence immunoassay for the detection of imidaclothiz was established by using magnetic nanoparticles (MNPs) as concentration elements and upconversion nanoparticles (UCNPs) as signal labels. The NaYF4/Yb,Er UCNPs and MNPs were conjugated with imidaclothiz monoclonal antibody and imidaclothiz antigen, respectively. Imidaclothiz could compete with the antigenconjugated MNPs for binding to the antibody-conjugated UCNPs and resulted in a decreased fluorescence signal when the MNPs were separated by an external magnet. Under the optimal conditions, the concentration of imidaclothiz producing 50% inhibition of the signal (IC50), limit of detection (LOD, IC10), and the linear assay range (IC10–IC90) were 14.59, 0.74, and 0.74–289.30 ng mL−1, respectively. The immunoassay exhibited no obvious cross-reactivity with analogues of imidaclothiz except for imidacloprid, with 89.2% cross-reactivity. The average recoveries measured in paddy water, pear, soil, peach, rice, tomato, wheat, and pakchoi were 75.7–105.2%, and the relative standard deviations (RSDs) were less than 11.2%. In addition, the results of the immunoassay correlated well with that of high-performance liquid chromatography (HPLC) for authentic samples.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-017-0653-7) contains supplementary material, which is available to authorized users. * Minghua Wang
[email protected] 1
College of Plant Protection (State & Local Joint Engineering Research Center of Green Pesticide Invention and Application), Nanjing Agricultural University, Nanjing 210095, China
2
Institute of Plant Protection, Jiangsu Academy of Agricultural Science, Nanjing 210014, China
Keywords Immunoassay . Imidaclothiz . Magnetic nanoparticles (MNPs) . Upconversion nanoparticles (UCNPs)
Introduction Imidaclothiz is one of the broad spectrum neonicotinoid insecticides that have been used widely to control sucking insects such as planthopper, aphid, whitefly, and leafhopper [1, 2]. Unfortunately, the overuse of neonicotinoid insecticides has caused environmental problems, including the harm to nontarget organisms such as honey bees and other pollinators [3]. In order to protect these pollinators, which are important to agricultural production, new methods must be available to rapidly detect imidaclothiz residues in environmental samples. Current methods of imidaclothiz detection rely heavily on instrumental analyses, including high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/ MS) [4, 5]. Although these methods have high accuracy and sensitivity, they consume large amounts of organic solvent and require complex procedures of sample preparation and purification [6]. Immunoassays, a simple, rapid, accurate, and efficient detection technology, have been widely used in the analysis of small molecules. In recent years, immunoassays that provide fluorescence readouts (e.g., fluorescence polarization immunoassay [7] and quantum dot fluorescent immunoassay [8]) have gained wide acceptance, since they are usually more sensitive than assays that rely on color changes. One of the drawbacks of these traditional fluorescent assays, however, is the excitation and emission spectra of their fluorophores, which are often subject to interferences, especially when they are used for analysis of agricultural products. Elimination of these interferences is often difficult [9]. The goal of this
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study was to establish simple and sensitive immunoassays for imidaclothiz that were not subject to the autofluorescence interference and photobleaching often encountered in studies that use traditional fluorophores. For the past few years, lanthanide-based upconversion nanoparticles (UCNPs), which emit short wavelength visible light when excited by long wavelength near-infrared (NIR) light, has attracted interest as a fluorophore that may provide a solution to this problem [10]. Compared with the traditional fluorescent material, UCNPs have many advantages, including stable physical and chemical properties and resistance to photobleaching. More importantly, UCNPs exhibit no background fluorescence interference, because the near-infrared excitation light is not absorbed by sample matrix; thus, the detection sensitivities of immunoassays are often improved when UCNPs are used as labeling material [11, 12]. Meanwhile, NH2–Fe3O4 magnetic nanoparticles (MNPs) with great water dispersion and rapid separation enrichment ability by the magnetic field that have widely used in immunoassay, cell separation, and biomedical applications [13–18]. In this work, we synthesized amine-functionalized NaYF4/ Yb,Er UCNPs and Fe3O4 MNPs and subsequently utilized these nanoparticles to create a competitive immunoassay for imidaclothiz. A rapid, simple, and high-sensitive competitive immunoassay for imidaclothiz was developed by using antibody-modified UCNPs as the detection probe and antigen-modified MNPs as the capture probe. The sensitivity, specificity, precision, and accuracy of the immunoassay were investigated. The results obtained with the immunoassay showed good correlation with results obtained from HPLC in environmental and agricultural samples.
MNPs were separated by a MS-12 magnetic separator (Alrun, China). Reagents FeCl3·6H2O, ethylene glycol, anhydrous sodium acetate, and 1,6-hexamethylenediamine were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA, 98.0%) was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China), 25% glutaraldehyde solution was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The antigen and monoclonal antibody of imidaclothiz (1E7) [19] and amino functional UCNPs [20] were prepared as previously described and stored in the laboratory. All other chemicals used in this study were analytically pure. Synthesis of amine-functionalized Fe3O4 MNPs The prepared of amine-functionalized Fe3O4 MNPs were based on the methods described by Wang and Li [21]. 1,6Hexamethylenediamine (6.5 g), anhydrous sodium acetate (2.0 g), and FeCl3·6H2O (1.0 g) were added into 30 mL ethylene glycol with stirring to form a homogeneous colloidal solution under the 50 °C. The above homogeneous colloidal solution was transferred into a 100-mL teflon-lined autoclave and heated to 196 °C for 6 h. After cooling to room temperature, the MNPs were separated using a magnetic separator. The separated black MNPs were washed three times first in 30 mL ultrapure water and then in 30 mL ethanol using the ultrasonic bath. The amino-functionalized Fe3O4 MNPs were subsequently dried at 60 °C overnight and then stored at 4 °C. Preparation of antibody-conjugated UCNPs and antigen-conjugated MNPs
Experimental Instrument The dimension and morphological characteristics of the MNPs were detected by an H-7650 transmission electron microscope (TEM, Hitachi, Japan). Fluorescence intensity was determined using an F-2700 fluorescence photometer (Hitachi, Japan) with an external 980-nm laser source (Changchun Laser Optoelectronics Technology Co., Ltd., China). The Fourier transform infrared spectrum (FTIR) and the phase composition of amine-functionalized MNPs were determined by a Vector-22 FTIR spectrophotometer (Bruker, Germany) and a D8 Advance X-ray diffraction (XRD, Bruker, Germany), respectively. The suspension liquid of MNPs was prepared in an ultrasonic bath (CQ-Ningbo Jiangnan Instrument Factory, China) subsequently vortexed (VM03 U, Crystal Technology & Industries, Inc., USA). The
The imidaclothiz antibody-conjugated UCNPs were prepared according to classical glutaraldehyde cross-linking method [22]. Ten milligrams amine-functionalized UCNPs were dispersed into 5 mL phosphate buffer solution (PBS, pH 7.4) by ultrasonic bath for 30 min. Sodium borohydride (100 mg) and 25% glutaraldehyde (1.25 mL) were subsequently added, and the reaction was shaken tardily for 1 h at room temperature. The UCNPs were separated by centrifugation and washed three times with 20 mL of PBS. Subsequently, the UCNPs were dispersed into 5 mL PBS and 600 μg of purified imidaclothiz antibody (in 0.6 mL) was added. The mixture was slowly shaken at room temperature for 6 h. The antibody not coupled to the UCNPS was removed by centrifugation at 6000×g. After removal of the supernatant, nonspecific and unreacted sites of imidaclothiz antibody-conjugated UCNPs were blocked by the addition of 5 mL of 1% BSA solution (0.01 mol L−1 PBS, pH 7.4) and the reaction was shaken
Upconversion fluorescence immunoassay for imidaclothiz by magnetic nanoparticle separation
slowly for another 6 h. After washing three times with 20 mL of PBS, the imidaclothiz antibody-conjugated UCNPs were dispersed into 5 mL of 0.01 mol L−1 PBS and stored at 4 °C. The identical procedure was used for the conjugation of imidaclothiz antigen (hapten-BSA) and aminofunctionalized MNPs. The process of immunoassay based on UCNPs and MNPs The immunoassay was performed by a competitive format; imidaclothiz competed with the antigen-conjugated MNPs for binding to the antibody-conjugated UCNPs and resulted in a decreased fluorescence signal when the MNPs were separated by an external magnet (Fig. 1). Typically, 280 μL of the standard solution of imidaclothiz or sample solution and 80 μL imidaclothiz antigen-conjugated MNPs were added into a 1.5-mL centrifuge tube with vortex blending, then 200 μL imidaclothiz antibody-conjugated UCNPs (2 mg mL−1) was added into the mixture. After incubating at 37 °C for 60 min, the MNPs were separated by using a magnetic separator and washed three times by PBS. The MNPs were dispersed in 0.5 mL PBS for detection of the fluorescence intensity using an F-2700 fluorescence spectrophotometer. Optimization of the immunoassay The experimental parameters, including the dosage of nanophase materials, pH value, concentration of sodium ions, methanol content, and incubation time were studied. The immunoassay was performed by varying the volume of imidaclothiz antigen-conjugated MNPs from 20 to 140 μL, the pH from 5 to 9, the concentration of sodium ions from 0.1 to 0.4 mol L−1, the methanol content from 0 to 10%, and Fig. 1 Schematic diagram of the upconversion fluorescence immunoassay
incubation time from 20 to 100 min. The assessment was based on Fmax/IC50, and the high value was the most desirable. The standard curve was established through the changes of fluorescence intensity values ΔI (ΔI = I0–I; I0 and I represent the fluorescence intensity in the absence and presence of imidaclothiz, respectively) and the logarithm of imidaclothiz concentration. The 50% of the inhibition signal (IC50) was calculated by using the regression equation [23]. Cross-reactivities The specificity of the immunoassay was determined by the cross-reactivities (CR). The analogues of imidaclothiz (from 10 to 10,000 ng mL−1) were detected by the immunoassay. The CRs were estimated by the following formula [24]: CR = (IC50of imidaclothiz/IC50of analogue) × 100 % . Analysis of spiked samples The agricultural and environmental samples including paddy water, soil, pear, rice, peach, wheat, tomato, and pakchoi which were obtained in Nanjing were used to spiked recovery study. The paddy water (10 mL) were filtered and spiked with known concentration imidaclothiz (5, 20, and 100 ng mL−1) and determined directly by mixing with the 2× PBS. The other samples (10 g) were homogenized and spiked with known concentration imidaclothiz (10 to 500 ng mL−1), then extracted twice by 10 mL of PBS containing 20% methanol by vortex for 3 min and ultrasonic treatment for 15 min. After centrifugation at 4500 rpm for 5 min, the supernatants were transferred into volumetric flask and adjusted to 25 mL with PBS. The concentrations of imidaclothiz were detected by the immunoassay after appropriate dilution with optimized PBS
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[25]. Each spiked sample was stored overnight after spiking with imidaclothiz and performed in triplicate.
Practical application of the established immunoassay In order to verify the accuracy of the established immunoassay, paddy water and pear were collected in different times from farm in Nanjing after sprayed with unknown concentration of imidaclothiz 10% WP. The samples were detected by immunoassay and HPLC, simultaneously. For the immunoassay, extraction and analytical methods were based according to the spiked recovery study. For HPLC, smashed pear samples (20 g), 50 mL acetonitrile, and 5 mL water were added into a triangular flask and extracted by vigorous shaking for 1 h. After filtering, the solution was transferred to a 100-mL mixing cylinder with stopper (containing 5 g NaCl) for layering. The half of the upper organic phase was transferred to a Florence flask and evaporated to dryness using a rotary evaporator. The filtered paddy water samples (10 mL) were extracted by 30 mL acetonitrile in separating funnel (containing 5 g NaCl); the aqueous phase of substrate was extracted again by 20 mL acetonitrile. Finally, all organic phase were merged into a Florence flask and evaporated to dryness. The concentrated extracts were dissolved by 2 mL mobile phase of HPLC (methanol:water = 30:70, v/v) and detected by HPLC (Agilent 1260) with an Eclipse pluseC18 column (250 mm × 4.6 mm, 5 μm); the flow rate and Fig. 2 Characterization of the amine-functionalized MNPs, TEM image (a), XRD pattern (b), and FT-IR spectrum (c), and the picture of MNPs dispersed in water (d)
injection volume were 0.9 mL min−1 and 20 μL, respectively. The ultraviolet detector wavelength was 270 nm.
Results and discussion Characteristics of MNPs The identification result of TEM and XRD for MNPs are shown in Fig. 2A, B. The average diameters of MNPs were within 10 nm; the XRD spectrogram can be conveniently retrieved from Fe3O4 (JCPDS 82-1533). The absorption peaks of FTIR at 3463 and 1621 cm−1 belong to the stretching vibration and deformation vibration of N–H, respectively. And the absorption peaks at 2851 and 2927 cm−1 belong to the stretching vibration of C–H; all of this demonstrates that the amine-functionalized MNPs match with 1,6-hexadiamine, indicating the existence of the free amino groups on the MNPs (Fig. 2C). Besides, the MNPs are well dispersed in water and remain stable (Fig. 2D). Optimization of the immunoassay The amount of antigen and antibody-conjugated nanoparticles (NPs) would influence the fluorescence intensity and the sensitivity of the immunoassay. In addition, the pH value, concentration of Na+, methanol content, and incubation time of
Upconversion fluorescence immunoassay for imidaclothiz by magnetic nanoparticle separation Fig. 3 The changes of fluorescence intensity with different concentrations of imidaclothiz (a) and the calibration curve of the immunoassay for imidaclothiz (b)
reaction system could also influence the sensitivity. The Electronic supplementary material (ESM) Fig. S1 shows the change of fluorescence intensity with variable volumes of
Table 1
MNPs (2 mg mL−1) (200 μL of 2 mg mL −1 antibodyUCNPs were used). When the volume of MNPs reached 80 μL, the maximal fluorescence intensity was observed,
Cross-reactivity of imidaclothiz toward its analogues
IC50 (ng mL-1)
CR (%)
Imidaclothiz
14.59
100
Imidacloprid
16.36
89.2
Compound
Chemical structure
Thiamethoxam
100,000
0.02
Nitenpyram
100,000
0.02
Thiacloprid
843.35
1.7
Acetamiprid
1540.20
1.0
Dinotefuran
100,000
0.02
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therefore, the 80 μL of MNPs was selected for subsequent experiments. ESM Fig. S2a–c shows that the IC50 and Fmax/ IC50 values with the variable pH, concentration of Na+ and methanol content, the pH 7.4, 0.2 mol L−1 concentration of Na+, and 2.5% methanol content were selected as the optimal conditions because the highest Fmax/IC50 value was obtained using these conditions. ESM Fig. S2d shows the changes of fluorescence intensity with different incubation times and that the fluorescence intensity tends to be stable when the incubation is carried out for 60 min.
87.50 ng mL−1) [19], fluorescence polarization immunoassay (FPIA, IC50 87.94 ng mL−1) [27], inner filter effect-based immunoassay (50% saturation of the signal (SC 50 ), 18.9 ng mL − 1 ) [20], and direct quantum dot-based fluoroimmunoassay (QDFIA, IC50 20.41 ng mL−1) [28], but it is inferior to time-resolved fluoroimmunoassay (TRFIA, IC50 6.91 ng mL−1) [28]. However, TRFIA is subject to interference by the Eu3+ in environment and has more operating steps (five steps) [29, 30], which have restricted its application.
Sensitivity Specificity Under optimal condition, the standard curve of the immunoassay was established by the fluorescence intensity change value ΔI (ordinate) and the logarithm value of concentration of imidaclothiz (abscissa) (Fig. 3). The IC50, limit of detection (LOD, IC10), and the linearity range (IC10–IC90) were 14.59, 0.74, and 0.74–289.30 ng mL−1, respectively. Compared with the reported immunoassays for imidaclothiz, this immunoassay has better sensitivity than the direct competitive enzymelinked immunosorbent assay (dc-ELISA, IC50 58.17 ng mL−1) [26], indirect competitive ELISA (ic-ELISA, IC 5 0 Table 2 Average recoveries of samples spiked with imidaclothiz
Sample
The CRs for the analogues were tested and shown in Table 1. The greatest CR value was found to be imidacloprid (89.2%), while others exhibited no significant CR (less than or equal to 1.73%). This is because imidaclothiz and imidacloprid have a similar imidazole ring and =N–NO2, which may play an important role in antigen-antibody reaction. The results were consistent with the reported immoassays [19, 26–28], which was decided by the nature of the anti-imidaclothiz antibody.
Spiked (ng g−1 or ng mL−1)
Measured ± SD (ng g−1 or ng mL−1)
Average recovery (%)
RSD (%)
Paddy water
5
5.1 ± 0.1
102.0
2.0
Soil
20 100 10 100 500
16.9 ± 1.9 88.5 ± 5.2 8.4 ± 0.9 88.2 ± 2.7 476.4 ± 9.8
84.5 88.5 83.7 88.2 95.3
11.2 5.8 10.8 3.1 2.0
10 100 500 10 100 500 50 200 500 50 200 500 50
8.8 ± 0.5 93.5 ± 2.1 479.2 ± 4.3 7.6 ± 0.8 105.2 ± 1.7 468.9 ± 14.0 41.1 ± 3.8 182.8 ± 6.6 475.9 ± 8.8 40.9 ± 1.5 163.8 ± 8.4 420.8 ± 16.0 45.6 ± 2.9
88.0 93.5 95.8 75.7 105.2 93.8 82.2 91.4 95.2 81.9 81.9 84.2 91.3
5.3 2.1 0.9 10.4 1.6 2.8 9.2 3.6 1.8 3.7 5.1 3.8 6.4
200 500 50 200 500
181.9 ± 5.8 505.2 ± 12.1 41.9 ± 3.2 173.4 ± 9.0 442.0 ± 17.1
91.0 101.0 83.8 86.7 88.4
2.9 2.4 7.6 5.2 3.9
Pear
Peach
Wheat
Tomato
Rice
Pakchoi
Upconversion fluorescence immunoassay for imidaclothiz by magnetic nanoparticle separation
Matrix effects The matrix in samples can influence the immunoreaction, and the dilution method with buffers is a simple and common way to eliminate the matrix effects in immunoassay. Paddy water, soil, pear, rice, peach, wheat, tomato, and pakchoi were used to explore the matrix effects. All samples were diluted to 2-, 5-, 10-, and 20-fold by PBS, respectively. The matrix effects were confirmed through comparing the imidaclothiz standard curves in matrix extracts and matrix-free 2.5% methanol-PBS solution. As shown in ESM Fig. S3, the 2-fold dilution for paddy water, 10-fold dilution for soil, pear, and peach, and 20fold dilution for rice, wheat, tomato, and pakchoi could eliminate the interference. Analysis of spiked sample Paddy water, soil, pear, rice, peach, wheat, tomato, and pakchoi samples spiked with known concentrations of imidaclothiz were extracted and detected as above. The results of the recoveries and RSDs are exhibited in Table 2. The acceptable recoveries were from 75.7 to 105.2%, and the RSDs were from 0.9 to 11.2%, which indicated that the great accuracy and precision of the immunoassay can meet the requirement for determination of residual imidaclothiz in agricultural and environmental samples. Correlation of the immunoassay with HPLC
different. The result indicated that the imidaclothiz in authentic samples could be simply, rapidly, and accurately detected by the developed immunoassay.
Conclusions A sensitive, rapid, and simple competitive fluorescence immunoassay for detection of imidaclothiz in agricultural and environmental samples was successfully developed. The method that combined the advantages of MNPs and UCNPs has a LOD of 0.74 ng mL−1 and IC50 of 14.59 ng mL−1, which was enhanced 6-fold compared with ic-ELSA [19] and FPIA [27] and 4-fold with dc-ELISA [26]. The detection of imidaclothiz by the method can be achieved through two steps in 60 min. In addition, the immunoassay results for the authentic samples were in great correlation with those of HPLC (y = 1.0322x − 0.8721, r = 0.9958). Due to the strong fluorescence signal that emitted from UCNPs was excited by a circumscribed 980 nm NIR laser, the interference of autofluorescence from the samples was eliminated. These results suggest that the UCNP and MNP labeling-based competitive fluorescent immunoassay could be ideally suited as a sensitive and quantitative method for detection of residual imidaclothiz in environmental and agricultural samples. Funding information This work was supported by the National Natural Science Foundation of China (31301690) and the National Key Research and Development Program of China (2016YFD0200207). Compliance with ethical standards
The authentic samples of paddy water and pear were analyzed by the immunoassay and HPLC. As shown in Fig. 4, a great correlation was acquired between the results of the immunoassay (X) and HPLC (Y) with the linear regression equation of y = 1.0322x − 0.8721 and r = 0.9958. Significant difference between the immunoassay and HPLC was assessed using Student’s t test; a P value of 0.9127 (greater than 0.05) implied that the data of the two methods were not significantly
Conflict of interest The authors declare that they have no conflict of interest.
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6. Fig. 4 Correlation between the developed immunoassay and HPLC for the real samples
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