Upconversion Nanoparticles and Monodispersed Magnetic

May 2, 2016 - Based on a competitive immunoassay format, the detection limit of the proposed method for detecting SQX was 0.1 μg L–1 in buffer and ...
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Upconversion Nanoparticles and Monodispersed Magnetic Polystyrene Microsphere Based Fluorescence Immunoassay for the Detection of Sulfaquinoxaline in Animal-Derived Foods Gaoshuang Hu,† Wei Sheng,†,‡ Yan Zhang,†,‡ Junping Wang,†,‡ Xuening Wu,† and Shuo Wang*,†,‡ †

Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science and Technology, Tianjin 300457, China ‡ Tianjin Food Safety & Low Carbon Manufacturing Collaborative Innovation Center, Tianjin 300457, China S Supporting Information *

ABSTRACT: A novel fluorescence immunoassay for detecting sulfaquinoxaline (SQX) in animal-derived foods was developed using NaYF4:Yb/Tm upconversion nanoparticles (UCNPs) conjugated with antibodies as fluorescence signal probes, and monodisperse magnetic polystyrene microspheres (MMPMs) modified with coating antigen as immune-sensing capture probes for trapping and separating the signal probes. Based on a competitive immunoassay format, the detection limit of the proposed method for detecting SQX was 0.1 μg L−1 in buffer and 0.5 μg kg−1 in food samples. The recoveries of SQX in spiked samples ranged from 69.80 to 133.00%, with coefficients of variation of 0.24−25.06%. The extraction procedure was fast, simple, and environmentally friendly, requiring no organic solvents. In particular, milk samples can be analyzed directly after simple dilution. This method has appealing properties, such as sensitive fluorescence response, a simple and fast extraction procedure, and environmental friendliness, and could be applied to detecting SQX in animal-derived foods. KEYWORDS: SQX, UCNPs, MMPMs, fluorescence immunoassay, animal-derived foods

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INTRODUCTION Sulfonamides (SAs) constitute one of the most important types of antibiotics used in human and veterinary treatment because of their low cost and broad-spectrum activity against both bacteria and certain microorganisms.1−3 Nevertheless, the extensive usage of these drugs, which poses a serious contamination risk to animal-derived foods, has become a public health concern because they can cause allergies, carcinogenesis, and the formation of resistant bacteria in the human body.4,5 To ensure consumer safety, maximum residue limits (MRLs) of SAs in foods have been proposed. In the European Union and China, the total SA residue should not exceed 100 μg kg−1 in animal-derived foods, such as meat and milk.6,7 Moreover, Korea set a MRL of 100 μg kg−1 for the sum of the 14 SAs, such as sulfaquinoxaline (SQX), sulfamethoxypyridazine, and sulfachloropyrazine, in marine products.8 Effective analysis methods were established to detect traces of SA residues, such as microbiological assays,9 capillary electrophoresis,10,11 gas chromatography−tandem mass spectrometry,12 and high-performance liquid chromatography 13,14 coupled with mass spectrometry.15,16 However, because of these methods’ requirements of long analysis times, expensive instruments and professional operators, their application for analysis has been limited. Compared with other analytical methods, enzyme-linked immunosorbent assay (ELISA),17−19 which has the advantages of being cheap and having high sensitivity, is currently widely used, but requires enzymatic reactions resulting in relatively time-consuming sample pretreatment to eliminate matrix effects. The fluorescence-based immuno approach has attracted increasing attention in recent years. Traditional fluorescent © XXXX American Chemical Society

materials, including organic dyes, dye-doped nanomaterials, and quantum dots, have shown some advantages, such as tunability, high brightness, and ease of conjugation with proteins.20−22 Unfortunately, their use also involves some inherent obstacles, such as their biotoxicity and their unstable optical and chemical features.23,24 Alternatively, lanthanide-based upconversion nanoparticles (UCNPs) were selected as a candidate for ideal fluorescence biolabels and have been successfully used in bioanalytical and bioimaging studies because of their unique properties, such as low autofluorescence background, good photochemical stability, nonblinking and nonbleaching emission, deep penetration into biological samples, and relatively low toxicity.25−28 UCNPs as fluorescence labels have not yet been used for the analysis of SA residues in animal-derived products, although a few studies applying related techniques for detecting mycotoxins and bacteria26,29 have been reported recently. Here, we developed a novel and sensitive fluorescent immunoassay for the determination of SAs combined with the optical and magnetic properties of UCNPs and monodispersed magnetic polystyrene microspheres (MMPMs) using SQX as a model. Furthermore, the proposed method was successfully applied for the determination of SQX in animal-derived foods. Received: April 1, 2016 Revised: April 27, 2016 Accepted: May 1, 2016

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DOI: 10.1021/acs.jafc.6b01497 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. Schematic of the fluorescence immunoassay protocol.

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Synthesis and Surface Modification of NaYF4:Yb/Tm UCNPs. The synthesis of NaYF4:Yb/Tm UCNPs was conducted according to the method described in our previous work.33 First, 1 mM LnAc3 (Y:Yb:Tm = 78 mol %:18 mol %:2 mol %), 6 mL of OA, and 17 mL of ODE were mixed under agitation to form a homogeneous solution. After being degassed at 100 °C for 10 min, the solution was heated to 160 °C and maintained for 30 min under argon. Then, the solution was cooled to room temperature naturally. Subsequently, 10 mL of methanol solution containing NH4F (4.0 mM) and NaOH (2.5 mM) was added and stirred for 30 min. The solution was first heated to 80 °C to remove the methanol and then to 300 °C for 1 h under argon. The solution was cooled to room temperature, and then the nanoparticles were washed with ethanol and collected by centrifugation. For the surface modification of the NaYF4:Yb/Tm UCNPs, a mixture of PAA (0.5 g) and diethylene glycol (10 mL) was heated to 110 °C with vigorous stirring under argon. Then 2 mL of toluene solution containing 30 mg of OA-capped UCNPs was slowly injected into this solution and stirred vigorously for 15 min to accelerate the evaporation of toluene before heating the solution to 110 °C for 1 h. Next, the system was heated to 240 °C and maintained for another 1 h. After the solution had cooled to room temperature, an excess of the dilute hydrochloric acid aqueous solution (pH 4−5) was added to the mixture. A white powder was obtained via centrifugation and washed three times with pure water to remove excess PAA. Finally, the OAcapped UCNPs (OA-UCNPs) were converted into biocompatible PAA-capped UCNPs (PAA-UCNPs) using appropriate functional groups (−COOH) through a ligand-exchange process. In this work, all of the upconversion fluorescence spectra were measured using a F-4500 fluorescence spectrophotometer (Hitachi Co., Japan) modified with an external 1 W adjustable continuous wave 980 nm laser (Beijing Hi-Tech Optoelectronic Co., China) as the excitation source instead of the xenon lamp. Preparation of Fluorescence Signal Probes and ImmuneSensing Capture Probes. EDC/NHS-assisted standard procedures were employed according to the method described previously33,34with slight modifications to trigger the reaction between the free carboxylic acid groups on UCNPs and the amino-containing antibodies to prepare the fluorescence signal probes. Briefly, 5 mg of PAA-UCNPs was dispersed in 2 mL of 2-(N-morpholino)ethanesulfonic acid buffer (MES, 10 mM, pH 5.5) by ultrasonication for 20 min. Then EDC (4 mM) and NHS (10 mM) were added to activate the carboxylic acid groups on their surfaces. The mixture was incubated at 30 °C for 2 h with continuous stirring. After washing three times, the precipitate was

MATERIALS AND METHODS

Materials and Instrument. Oleic acid (OA, 90%) and 1octadecene (ODE, 90%) were obtained from Alfa Aesar Co. Ltd. (Ward Hill, MA, USA). C6H9O6Yb·4H2O (99.9%), C6H9Tm·xH2O (99.9%), poly(acrylic acid) PAA (M1/41800), and diethylene glycol (DEG) were purchased from Sigma-Aldrich Co. Ltd. (St. Louis, USA), as were the SA standards, N-hydroxysuccinimide (NHS), 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide (EDC), bovine serum albumin (BSA, molecular weight [MW] 67 kDa), and ovalbumin (OVA, MW 45 kDa). Y(OOCCH3)3·4H2O was purchased from Aladdin Industrial (Shanghai, China). The commercial ELISA kits were purchased from Reagent Technology Co. Ltd. (Shenzhen, China). Bicinchoninic acid (BCA) protein quantification kits were obtained from Solarbio Science and Technology Co., Ltd. (Beijing, China). MMPMs were purchased from Tianjin BaseLine ChromTech Research Centre (Tianjin, China). Anti-SQX monoclonal antibody (McAb) and SQX hapten were both produced in our laboratory. All other reagents used in this work were at least analytical grade and were purchased from Tianjin No.1 Chemical Reagent Factory (Tianjin, China). The solutions were prepared with deionized water from a Milli-Q system (Millipore, Billerica, MA, USA). The size and morphology of the nanoparticles were determined using a JEM-2010 FEF transmission electron microscope (TEM, JEOL Ltd., Japan) operated at 200 kV. Fourier transform infrared (FT-IR) spectra of the modified nanoparticles were obtained using a Nicolet Nexus 470 Fourier transform infrared spectrophotometer (Thermo Electron Co., USA) with the KBr method. Preparation of Antibody and Coating Antigen. The McAb against SQX was purified by the caprylic acid ammonium sulfate method30−32 and stored at 4 °C for further use. Identification of specific immunoglobulin was performed with a mouse mAb Isotyping Kit (Sigma-Aldrich, USA) according to the manufacturer’s protocol, and the isotype was identified as IgG2a alongwith kappa (κ) light chain. The coating antigen was prepared by the active ester method as described in the literature.30 Freshly prepared EDC·HCl (9.3 mg) was added to a hapten solution (0.025 mM hapten in 200 μL of anhydrous dimethylformamide). The reaction mixture was subsequently mixed with a solution of 10.0 mg of OVA dissolved in 2 mL of phosphatebuffered saline (PBS, 0.01 M, pH 7.4) and kept at room temperature for 4−6 h with continuous magnetic stirring. Then, another 4.7 mg of EDC·HCl was added. The mixture was stirred at 4 °C overnight, and the resulting solution was dialyzed against PBS (0.01 M, pH 7.4) for 3 days. Finally, the coating antigen (SQX-OVA, 3.75 g L−1) was obtained and stored at 4 °C for further use. B

DOI: 10.1021/acs.jafc.6b01497 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 2. Characterization of the materials: TEM images of OA-UCNPs (A) and PAA-UCNPs (B), fluorescence spectra (C), and FT-IR spectra (D) of OA-UCNPs and PAA-UCNPs, respectively. redispersed in 2 mL of N-(2-hydroxyethyl) piperazine-N-ethanesulfonic acid buffer (HEPES, 10 mM, pH 7.2) containing serial amounts of McAb against SQX. The linkage reaction was processed at 30 °C for 2 h. Subsequently, 15 mg of BSA was added to block any unreacted NHS and nonspecific sites. Finally, the resultant compounds were centrifuged, washed, and dispersed in 1 mL of HEPES buffer containing 0.1% BSA for further applications. Similarly, the classic active ester method was used to conjugate the coating antigen to the surface of MMPMs. First, to obtain carboxylactivated MMPMs, EDC and NHS were added to 5 mL of PBS buffer (0.01 M, pH 7.4) containing 5 mg of MMPMs with stirring for 2 h in a reciprocating oscillator at room temperature. After washing three times with PBS and magnetic separation in an external magnetic field, coating antigens (SQX-OVA) were added to the solution and stirred slowly for 4 h at room temperature. Next, SQX-OVA-MMPMs conjugates were blocked with 2% BSA. Finally, the prepared immunesensing capture probes were stored in 5 mL of PBS at 4 °C prior to use. Immunoassay Procedure. A schematic illustration of the fluorescence immunoassay for the detection of SQX is shown in Figure 1. Sensing probes (SQX-OVA-MMPMs) are used to capture signal probes (McAb-PAA-UCNPs), and with the assistance of a magnetic field, the resulting immunocomplexes (PAA-UCNPs-McAbNOR-OVA-MMPMs) can be easily separated from the unreacted substances. The number of the immunocomplexes decreased in the presence of free SQX, which could simultaneously react with signal probes in a competitive process, making the fluorescence signal gradually decrease. First, 100 μL of signal probes (McAb-PAA-UCNPs) was added to 1.5 mL centrifuge tubes containing 100 μL of the running concentrations of SQX standard solutions (or sample) and 100 μL of sensing probes (SQX-OVA-MMPMs). Then, the mixture was incubated for 1 h at room temperature with gentle shaking. The resulting mixture then was separated by an external magnet for 2 min and rinsed three times with PBS buffer (0.01 M, pH 7.4). Trapped particles were resuspended in 600 μL of PBS (0.01 M, pH 7.4), and the fluorescent emission intensity (recorded at 474 nm) was then assayed using the F-4500 fluorescence spectrophotometer equipped

with a 980 nm laser as the excitation source. The scan speed was 300 nm min−1. Sample Pretreatment. Milk, tissue samples (chicken, beef, and pork), and marine product samples (sea bass and shrimp), which were certified as free of SQX, were purchased from local supermarkets. The samples were fortified with SQX to final concentrations of 0.5, 5, 50, and 500 μg kg−1. The tissues and marine products samples were minced with a highspeed disintegrator. Next, 1 g aliquots of each sample were added to 50 mL centrifuge tubes and spiked with an appropriate SQX solution. Then, 2 mL of carbonate/hydrogen carbonate extraction buffer (pH 10, 0.2 M Na2CO3, 0.2 M NaHCO3, distilled water, 1.22:1:6.62; v/v/ v) was added. The mixture was vortexed vigorously and extracted for 5 min. Subsequently, the supernatant was collected after centrifugation of the homogenized samples (10,000 rpm, 5 min, 4 °C), and the pH was adjusted to 7−7.5 with 1 M HCl.35,36 Finally, the extracts were diluted with PBS at a ratio of 1:2.5, and the resulting solution was used for analysis. Milk samples were treated without any extraction; after 5-fold dilution with PBS (0.01 M, pH 7.4), they could be measured directly.

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RESULTS AND DISCUSSION UCNP Characterization. The prepared OA-UCNPs and PAA-UCNPs were characterized, as shown in Figure 2. The TEM images reveal that the morphology of the NaYF4:Yb/Tm UCNPs is characterized by well-dispersed and spherical nanoparticles with very smooth surfaces and average uniform diameters of about 30 and 40 nm before (Figure 2A) and after PAA-modification (Figure 2B), respectively. The size of the PAA-UCNPs increased because of the formation of a thin PAA layer on the surface of the bare UCNPs. Figure 2C shows the fluorescence spectra of OA-UCNPs and PAA-UCNPs excited with a 980 nm laser with 1 W output. The result indicates that no significant difference in fluorescence intensity existed between OA-UCNPs and PAA-UCNPs. The UCNPs obtained in this study showed strong fluorescence intensity and a single C

DOI: 10.1021/acs.jafc.6b01497 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry characteristic emission peak at 474 nm without disturbance peaks. The blue emission peak was assigned to the 1G4 → 3H6 transition.37 FT-IR spectra of OA-UCNPs and PAA-UCNPs are shown in Figure 2D. Two strong bands at approximately 1450 and 1562 cm−1 were observed for OA-UCNPs, which were assigned to the asymmetric and symmetric vibrations of the carboxylate anions on the surface of the OA-UCNPs. However, this feature was apparently lost after ligand exchange with PAA. Compared with the spectrum of OA-UCNPs, a new characteristic peak appeared at 1730 cm−1 in the spectrum of PAA-UCNPs, indicating the increasing quantity of −COOH groups on the particle surface. In addition, strong peaks at approximately 2927 and 2854 cm−1 were associated with the asymmetric and symmetric stretching vibration modes, respectively, of repetitive −CH2− groups of OA in the long alkyl chain. However, these peak intensities weakened after PAA exchange. The results demonstrated that the OA-UCNPs were converted into hydrophilic UCNPs by ligand exchange of OA with PAA, facilitating their easy dispersion in water, which is convenient for further applications. Characterization of Fluorescence Signal Probes and Immune-Sensing Probes. The conjugation amount of antibody and coating antigen on the surfaces of UCNPs and MMPMs was investigated using BCA protein quantitation kits. Varying amounts of McAb (20 μg to 120 μg) were added to 1 mL of PAA-UCNPs solution. The supernatant was collected after removing the obtained complex via centrifugation and testing it with the BCA protein quantitation kits. The amount of conjugated antibodies increased as the amount of McAb increased, but in terms of the conjugate efficiency and capacity, 80 μg of anti-SQX McAb was found to be optimal and used for preparing the signal probes, as shown in Figure 3A. For sensing probes, different amounts of coating antigen (5 μg to 55 μg) were added to 1 mL of MMPMs solution. The supernatant was collected after removal of the obtained complex via magnetic separation and tested with a BCA protein quantitation kit. The conjugation capacity of the MMPMs reached saturation when the amount of SQX-OVA was equal to or greater than 25 μg (Figure 3B). Because a larger amount of SQX-OVA caused a lower conjugation ratio and wasted reagents, 25 μg of SQX-OVA was finally selected. In a competitive immunoassay, excessive signal probes will not obviously alter fluorescence signals in the presence of low concentrations of SQX. Thus, careful optimization of this component is important to achieve the quantitative analysis of trace SQX. In our study, 100 μL of sensing probes (SQX-OVAMMPMs) was mixed with varying amounts of signal probes. Then, the mixture was incubated for 1 h at room temperature. Figure 3 indicates that the fluorescence intensity increases sharply as the amount of signal probes increases initially and then becomes saturated when the amount of signal probes reaches 100 μL. Therefore, 100 μL of signal probes was determined to result in the best fluorescence signal performance in this study. Analysis Procedure. Under the optimized conditions, a facile method to detect SQX was performed. The fluorescence intensity obtained was assayed using the F-4500 fluorescence spectrophotometer equipped with the 980 nm laser, and 474 nm was chosen as the detection wavelength. To evaluate the detection limit of the fluorescence immunoassay, different concentrations of SQX were analyzed (0, 0.5, 1.0, 5.0, 10, 50, and 100 μg L−1). The fluorescence

Figure 3. Effect of fluorescence signal probes on the fluorescence immunoassay. (A) Optimization of the amount of antibody bound to PAA-UCNPs to prepare signal probes; the conjugation rate was defined as the ratio of the decreased absorbance of the supernatant solution to the absorbance of the original antibody solution using BCA kits. (B) Optimization of the amount of coating antigen bound to the surface of MMPMs to prepare sensing probes; the conjugation rate was defined as the ratio of the decreased absorbance of the supernatant solution to the absorbance of the original coating antigen solution (SQX-OVA) using BCA kits. (C) Fluorescence intensity recorded with various amounts of signal probes after incubation with 100 μL of sensing probes for 1 h. Each data point is the mean of triplicate analyses.

intensity was maximized in the absence of SQX and decreased with increasing SQX concentration. Thus, the concentration of SQX was proportional to the decreased fluorescence intensity. The standard curve was plotted, as shown in Figure 4. The linear range spanned from 0.1 to 100 μg L−1 (R2 = 0.9905), and the detection limit of the proposed method for SQX was 0.1 μg L−1. Specificity. Specificity was investigated by assessing the fluorescence signal change value between SQX and the other SAs analogues (10 μg L−1). The results are shown in Figure 5; sulfachloropyridazine, sulfamethoxazole, and sulfamethoxypyridazine induced relatively strong fluorescence signal changes compared with SQX, but others did not. As a conclusion, the established fluorescence immunoassay could be applied to detecting 4 types of SAs because of the combination property of the antibody used in this experiment. Sample Analysis. To confirm the efficiency of this method for the detection of SQX, animal-derived foods (milk, tissue samples, and marine product samples) spiked with SQX at four levels were analyzed using the proposed fluorescence immunoassay. The recoveries of SQX, as shown in Table 1, ranged from 69.80% to 133.00%, with CV values of 0.49%−25.99%, indicating the good accuracy and precision of this method. Immunoassay Validation. Spiked SQX samples were analyzed by commercial ELISA kits to verify the practicability of the fluorescence immunoassay. As shown in Table 2, the results are in good agreement with those of the commercial ELISA kits. For the milk samples, the fluorescence immunoassay has a 10-fold-lower detection limit than the commercial ELISA kits. A comparison of analysis procedures between this method and commercial ELISA kits for detecting SQX in animal-derived foods was performed and is shown in Figure 6. For tissue and marine samples, the sample-extraction D

DOI: 10.1021/acs.jafc.6b01497 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Table 1. Recoveries and Coefficient of Variation (CV) Values for SQX in Animal-Derived Foods by the Proposed Method (n = 3) intra-assayc samples chickena

beefa

porka

Figure 4. Standard curve of the fluorescence immunoassay for SQX in PBS buffer. The fluorescence intensity was maximized in the absence of SQX, and that of the immune complexes (PAA-UCNPs-McAbSQX-OVA-MMPMs) obtained after magnetic separation decreased as the SQX concentration increased. The inset equation represents the linear relationship between the concentration of SQX and the variations in the fluorescence intensity (ΔI). Each data point is the mean of triplicate analyses.

shrimpa

sea bassa

milkb

interassayd

spiked concn

recovery (%)

CV (%)

recovery (%)

CV (%)

0.5 5 50 500 0.5 5 50 500 0.5 5 50 500 0.5 5 50 500 0.5 5 50 500 0.5 5 50 500

82.35 86.31 69.80 92.98 133.00 97.07 101.08 78.85 112.14 117.59 101.27 95.77 97.11 108.28 96.03 111.36 80.34 109.36 101.69 80.96 108.49 85.78 97.60 102.91

18.63 4.61 0.49 10.07 7.52 10.42 11.32 3.20 15.41 10.84 9.15 1.39 22.52 8.74 7.48 16.71 20.08 22.54 13.04 8.08 9.79 2.87 2.03 14.00

77.40 129.00 93.83 120.62 102.32 105.88 92.41 102.77 117.32 95.52 121.70 89.11 102.91 111.41 109.83 100.44 96.83 109.12 119.92 91.96 99.46 82.46 99.91 119.93

15.63 11.21 18.90 19.19 18.53 25.99 17.79 16.94 11.43 21.63 2.44 20.38 24.40 22.11 8.59 16.05 17.67 6.51 16.84 24.23 10.39 7.87 2.18 15.23

a

The concentration unit used for chicken, beef, pork, shrimp, and squid samples is μg kg−1. bThe concentration unit used for milk samples is μg L−1. cIntra-assay variations were determined by three replicates on a single day. dInterassay variations were determined by a single test on three different days.

Table 2. Verification of the Proposed Method for SQX Detection in Real Samples Using a Commercial ELISA Kit (n = 3) detected concn (mean ± SD) samples porka

Figure 5. Specificity analysis of the proposed fluorescence immunoassay for SAs in PBS buffer. Each data point is the mean of triplicate analyses.

shrimpa

procedures were simple and the total pretreatment time was 10−15 min using the proposed method, however the total pretreatment time was approximately 1 h because an extended period of sample drying under blowing nitrogen gas was needed according to the protocol using commercial ELISA kits. For milk sample, it could be analyzed directly after dilution without centrifugation using this proposed method. This method with simple sample-extraction procedure showed good performance and excellent resistance to interference from the food matrix in our study. These features might be because of the following two reasons. One is the unique luminescence property of UCNPs that emits shorter wavelength (ultraviolet or visible) light under excitation by longer wavelength (usually near-infrared) light via

milkb

spiked concn

this method

ELISA kit

0.5 5 50 500 0.5 5 50 500 0.5 5 50 500

0.46 ± 0.07 5.88 ± 0.73 40.64 ± 1.05 448.83 ± 44.37 0.49 ± 0.11 5.41 ± 0.47 48.01 ± 3.59 516.79 ± 93.06 0.54 ± 0.05 4.29 ± 0.12 48.80 ± 0.99 514.57 ± 90.73

0.43 ± 0.04 5.17 ± 0.20 42.15 ± 1.27 445.48 ± 46.58 0.55 ± 0.06 5.55 ± 0.35 47.94 ± 0.56 523.06 ± 21.26 ndc 4.36 ± 0.40 44.38 ± 1.30 455.79 ± 25.22

a

The concentration unit used for chicken, beef, pork, shrimp, and squid samples is μg kg−1. bThe concentration unit used for milk samples is μg L−1. cNot detected. The concentration is lower than the detection limit of the ELISA kit.

multiphoton absorption mechanism,27 which efficiently prevents autofluorescence originating from biomolecules possibly contained in solution, allowing excellent resistance to interference from the food matrix. Then the use of MMPMs, E

DOI: 10.1021/acs.jafc.6b01497 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 6. Comparison of analysis procedures between this method and commercial ELISA kits for detecting SQX in animal-derived foods.

no organic solvent, used in the experiment was more environmentally friendly. Furthermore, the established immunoassay showed satisfactory sensitivity (0.5 μg kg−1 in samples). In particular, the detection limit for the milk samples was more sensitive than that of the commercial ELISA kits. These results suggest that the established fluorescence immunoassay has advantages of sensitive fluorescence response, a simple and fast operation procedure, environmental friendliness, and excellent resistance to interference from the food matrix. Thus, this method is applicable for the rapid and accurate detection of SQX in animal-derived foods. We believe that such a UCNPbased fluorescent immunoassay will have great potential applications for determination of contaminants or foodborne toxicants in food safety fields. Furthermore, this method could be extended to the simultaneous detection of multiple targets in a single analysis system based on the multicolor labeling character of UCNPs in the future.

which maintain their inherent magnetic properties and low toxicity but show better dispersion in solution than Fe3O4 magnetic nanoparticles and can be isolated readily from sample matrices using an external magnet, favors capturing fluorescent signal probes and achieving high extraction efficiency in the comparative immunoassay. The method using an external magnet without centrifugation was time-saving. Besides, it is worth mentioning that the extraction buffer without organic solvent used in the experiment was relatively environmentally friendly. In summary, the use of UCNPs, which were synthesized by the addition of different types and ratios of the lanthanide (Tm) and showed emission at 474 nm with bright blue luminescence, for a fluorescence-based immunoassay using magnetic separation to detect SQX in animal-derived foods was proposed for the first time. The use of UCNPs, which have unique luminescence properties, such as lack of autofluorescence and a light scattering background, enhanced the performance of the established method regarding its resistance to interference from the food matrix. In addition, the use of MMPMs, which showed better dispersion in solution than Fe3O4 magnetic nanoparticles, facilitated the separation of the immunocomplex from the mixed system without centrifugation, thereby greatly simplifying the overall operation procedure. The fluorescence immunoassay was accurate for the detection of SQX residues in food samples, and the results were in good agreement with those from commercial ELISA kits. Milk samples could be analyzed directly with a 5-fold dilution by PBS, and tissue or marine samples could be analyzed after fast extraction procedures compared with those required for commercial ELISA kits. The extraction buffer, which contained

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b01497.

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Schemes of preparing fluorescence signal probes and immune-sensing probes (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel: +86 22 6091 2483. Fax: +86 22 6091 2493. E-mail: s. [email protected]. F

DOI: 10.1021/acs.jafc.6b01497 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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The authors are grateful for financial support from International Science and Technology Cooperation Program of China (Project No. 2014DFR30350), the National Natural Science Foundation of China (Project No. 31201353), the Tianjin Municipal Science and Technology Commission (Project No. 13JCQNJC15300), and the Ministry of Science and Technology of the People’s Republic of China (Project No. 2012BAD28B05). Notes

The authors declare no competing financial interest. This article does not contain any studies with laboratory animals or human participants performed by any of the authors.

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ABBREVIATIONS USED BSA, bovine serum albumin; EDC, 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide; MRL, maximum residue limit; MMPMs, monodispersed magnetic polystyrene microspheres; NHS, N-hydroxysuccinimide; OVA, ovalbumin; OA, oleic acid; ODE, 1-octadecene; PAA, poly(acrylic acid); SAs, sulfonamides; SQX, sulfaquinoxaline; UCNPs, upconversion nanoparticles

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DOI: 10.1021/acs.jafc.6b01497 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.6b01497 J. Agric. Food Chem. XXXX, XXX, XXX−XXX