Magnetic Quantum Dot Nanobead-Based Fluorescent

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Magnetic quantum dot nanobead-based fluorescent immunochromatographic assay for the highly sensitive detection of aflatoxin B1 in dark soy sauce Liang Guo, Yanna Shao, Hong Duan, Wei Ma, Yuankui Leng, Xiaolin Huang, and Yonghua Xiong Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00223 • Publication Date (Web): 06 Mar 2019 Downloaded from http://pubs.acs.org on March 8, 2019

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Analytical Chemistry

Magnetic quantum dot nanobead-based fluorescent immunochromatographic assay for the highly sensitive detection of aflatoxin B1 in dark soy sauce

Liang Guoa,b#, Yanna Shaoa#, Hong Duana,b, Wei Maa,c, Yuankui Lenga, Xiaolin Huang*a, and Yonghua Xiong*a,b

a

State Key Laboratory of Food Science and Technology, Nanchang University,

Nanchang 330047, P. R. China; b

Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, P. R.

China; c

Gaoping Center for Comprehensive Inspection and Testing, Gaoping 048411, P. R.

China #

These authors contributed equally to this work

*Correspondence

to:

Dr. Xiaolin Huang and Dr. Yonghua Xiong State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, P.R. China E-mail: [email protected]; [email protected]

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Herein, we synthesized bifunctional magnetic fluorescent beads (MFBs) with a distinct core/shell structure by encapsulating octadecylamine-coated CdSe/ZnS QDs (OC-QDs) and oleic acid-modified iron oxide nanoparticles (OA-IONPs) into two polymer matrixes with different hydrophobic properties. The OC-QDs and OA-IONPs were mainly distributed in the outer layer of MFBs. The resultant MFBs displayed ca. 226-fold stronger fluorescence emission relative to the corresponding OC-QDs and retained ca. 45.4% of the saturation magnetization of the OA-IONPs. The MFBs were used to purify and enrich aflatoxin B1 (AFB1) from dark soy sauce and then utilized as a fluorescent reporter of immunochromatographic assay (ICA) for the sensitive detection of AFB1. Under the optimal detection conditions, the MFB-based ICA (MFB-ICA) displayed a dynamic linear detection of AFB1 in sauce extract over the range of 5 pg/mL to 150 pg/mL with a half maximal inhibitory concentration of 27 ± 3 pg/mL (n = 3). The detection limits for AFB1 in sauce extract and real dark soy sauce were 3 and 49 pg/mL, respectively, which are considerably better than those of previously reported fluorescent bead-based ICA methods. The analytical performance of the proposed MFB-ICA in terms of selectivity and accuracy was investigated by analyzing AFB1-spiked dark soy sauce samples. The reliability of the proposed method was further confirmed by ultra-performance liquid chromatography with fluorescence detection. With the combined advantages of QDs and IONPs, the resultant MFBs offer great potential as reporters of ICA for the sensitive detection of trace pollutants in complex matrix samples.


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Keywords: magnetic fluorescent beads; immunochromatographic assay; aflatoxin B1; dark soy sauce



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INTRODUCTION Membrane-based immunochromatographic assay (ICA) is a widely used point-of-care testing (POCT) technology in environmental monitoring,1,2 food analysis,3, and in vitro diagnosis4,5 because of its simplicity, rapidity, portability, cost-effectiveness, and user-friendliness.6-8 However, the detection of ultra-trace analytes in complex samples remains challenging. The matrix interferences of complex samples and insufficient analytes due to the limited transferred sample fluid are major factors decreasing the sensitivity of ICA.4 Specific preconcentration of analytes based on immunomagnetic separation (IMS) is a simple and feasible strategy to overcome these challenges and improve the detection sensitivity of ICA. This strategy has been widely used for the detection of analytes, including microbes,9-12 virus,13,14 proteins,15 and small molecules. However, the output signals of magnetic material-based competitive ICAs for small molecules are limited to magnetic16 and colorimetric (gray value of magnetic particles) signals,17-20 providing limited detection sensitivity. Recently, various fluorescence materials have been proposed as reporters to enhance the sensitivity of ICA because the fluorescence signal is inherently more sensitive than the colorimetric signal of traditional colloidal gold and magnetic nanoparticles (MNPs). Among these fluorescence materials, quantum dots (QDs) are promising reporters for developing highly sensitive ICA because of their unique fluorescent properties, such as size-tunable emission, broad adsorption, narrow and symmetric

photoluminescence

spectrum,

strong


luminescence,

and

robust

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photo-stability. Moreover, polymer nanobead-encapsulated numerous quantum dots (QBs) can provide higher detection sensitivity for ICA than single QDs.22-24 Therefore, QBs are promising labels for developing highly sensitive ICA. In consideration of the distinct but complementary advantages of QBs and magnetic nanobeads, using magnetic fluorescent beads (MFBs) as ICA reporters could markedly improve the sensitivity and accuracy for target analytes in complex samples. Previous studies prepared difunctional QD/MNP composites by using various techniques, including in-situ growth strategy,25,26 layer-by-layer assembly,27,28 template-based embedment,29 and emulsion technique.30 Emulsion techniques for incorporating QDs and MNPs into polymer beads have attracted considerable attention because of their simplicity and versatility. Given their potential and versatility in generating monodisperse emulsion droplets, microfluidic techniques have been applied to fabricate QD/MNP composite microspheres.31-33 However, the resulting microspheres show micron-level size, rendering them unsuitable for application in ICA.23 By contrast, ultrasonic emulsification can be used to prepare emulsion conveniently with tunable sizes ranging from nano-level to micron-level34-37 and has been proposed to prepare QBs as reporters of ICA.22-24 In the present study, highly luminescent MFBs were fabricated via a facile one-pot ultrasonic emulsification by encapsulating numerous octadecylamine-coated CdSe/ZnS QDs (OC-QDs) and oleic acid-modified iron oxide nanoparticles (OA-IONPs)

into

poly(methyl

methacrylate)

(PMMA)

and

poly(maleic

anhydride-alt-1-octadecene) (PMAO) composites. The as-prepared MFBs showed a



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distinct core/shell structure because of the hydrophobic difference of OC-QDs, OA-IONPs, PMAO, and PMMA, where QDs and IONPs were mainly distributed in the outer layer of MFBs. As a result, the MFBs retained ca. 45.4% of the saturation magnetization compared with the OA-IONPs and displayed ca. 226-fold stronger fluorescence emission relative to the corresponding QDs. The resultant MFBs were then used as an ICA reporter for the detection of trace aflatoxin B1 (AFB1) in dark soy sauce. Soy sauce, classified as dark or light, is a basic condiment in Asia. It is made from fermenting boiled soybeans and roasted grain that are possibly contaminated with AFB1.38 Various analytical methods, including high-performance liquid chromatography (HPLC),39 liquid chromatography-mass spectrometry (LC-MS),40 enzyme-linked immunosorbent assay (ELISA),41,42 immunobiosensors,

43

and ICA

methods44 have been reported for AFB1 determination. Among them, ICA strip is one of the most popular on-site screening methods. However, the dark soy sauce is thicker and darker in color than light soy sauce because of its greater amounts of pigment components, which cause matrix interferences to ICA. To improve the sensitivity and accuracy of ICA, we used anti-AFB1 antibody-labeled MFBs to enrich AFB1 molecules and remove the interfering components in dark soy sauce samples. Owing to the preconcentration and purification via IMS, the proposed MFB-based competitive ICA shows an obviously enhanced detection performance with respect to sensitivity, accuracy, and robustness for trace AFB1 detection in dark soy source samples. To our best knowledge, the usage of MFBs as a bifuctional probe for simultaneously preconcentrating target and amplifying signal of competitive ICA


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sensor has not been reported.

Scheme 1. (A) Schematic illustration of the MFBs formation. (B) The principle of the MFBs based ICA.



EXPERIMENTAL SECTION Materials and Reagents. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl), PMMA, PMAO, FeSO4·7H2O, FeCl3·6H2O, oleic acid, AFB1, and bovine serum albumin (BSA) were provided by Sigma–Aldrich Chemical (St. Louis, MO). AFB1–BSA conjugates (molar ratio of 15:1) and unpurified ascites containing anti-AFB1 monoclonal antibodies (anti-AFB1 mAbs) were synthesized in our laboratory. Donkey anti-mouse IgG was provided by Beijing Zhongshan Biotechnology, Inc. (Beijing, China). The sample pad, nitrocellulose membrane (NC, CN95), and absorbent pad were purchased from Schleicher and Schuell GmbH (Dassel, Germany). All chemicals were of analytical grade or better and obtained from Sinopharm Chemical Corp. (Shanghai, China). Apparatus. The ultrasonicator (II D) was purchased from Ningbo scientz biotechnology Co., Ltd. (Ningbo, China). The BioDot XYZ platform equipped with a motion controller, BioJet Quanti 3000k dispenser, and AirJet Quanti 3000k dispenser, were obtained by BioDot (Irvine, CA). The automatic programmable cutter was provided by Shanghai Jinbiao Biotechnology Co., Ltd. (Shanghai, China). The fluorescent strip reader was purchased from Suzhou Hemai Precision Instrument Co., Ltd. (Jiangsu, China). Milli-Q water was applied in this work (Molsheim, France). Preparation of MFBs. OC-QDs and OA-IONPs were synthesized as previously described.22,29 The carboxyl group-modified MFBs were prepared by incorporating OC-QDs and OA-IONPs into polymer nanobeads via an ultrasonic emulsification technique. In brief, 8 mg of OC-QDs, 6 mg of OA-IONPs, 20 mg of PMMA, and 17


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mg of PMAO were completely dissolved in 0.6 mL of trichloromethane to form an oil phase, which was then added into 1 mL of SDS solution (2.5 mg/mL) to form a mixed solution of oil and water. Then, the mini-emulsion was prepared via a 2 min ultrasonic emulsification process using an ultrasonicator (working, 10 s; pausing, 5 s) at 104.5 W. Then, MFBs were obtained after a 4 h evaporation process of trichloromethane at 45 °C. The resulting MFBs were washed with PBS via centrifugation, and the carboxyl group on the surface of the MFBs was obtained by hydrolyzing the anhydride groups of PMAO in water for 24 h. The size distribution and zeta potential of the MFBs were measured using a Zetasizer Nano instrument (ZEN3700, Malvern). The morphology and structure were characterized by a high-resolution TEM (JEOL JEM 2100). The fluorescence spectra of the as-prepared MFBs were collected on an F-4500 fluorescence spectrophotometer (Hitachi Ltd). Preparation of the MFB-mAbs. The MFB-mAbs were synthesized through the EDC conjugation method as earlier reported.24 In brief, the carboxyl groups of MFBs were activated by adding 0.1 mg of MFBs into 2.7 mL of 0.01 M PB buffer solution (pH 6.0) containing 0.1 μg of EDC. After activation, the desired amounts of unpurified ascites containing anti-AFB1 mAbs were added drop-by-drop into the above mixed solution under gentle stirring. After reaction for another 40 min at 25 ℃, the resulting MFB-mAbs were washed with PBS via centrifugation at 13,500 g for 10 min. The as-prepared MFB-mAbs were re-suspended in 1 mL of home-made probe storage solution24 and then conserved at 4 °C for further use. Fabrication of MFB-ICA Sensor. The MFB-ICA strip was constructed according to



our previous work with slight modification.22 In this case, 0.5 mg/mL of AFB1-BSA conjugates and 1 mg/mL of donkey anti-mouse IgG were respectively sprayed onto the NC membrane as the Test line (T line) and Control line (C line) at a density of 0.4 μL/cm, and then the NC membrane was stored at 37 °C for drying overnight. The other operations were conducted following our previously described protocol. Quantitative Procedure of MFB-ICA Sensor. A 5.0 μL aliquot of MFB-mAbs (50

μg/mL) was incubated with 350 μL of the sample solution at 37 °C for 20 min. Then, the MFB-mAbs and analyte immunocomplexes were collected by using an external magnetic field and then re-suspended in 70 μL of PBS–ethanol mixture (95:5, v/v). The suspension was added into the well of the sample pad, and the strip was scanned with a fluorescence strip reader after a 15 min running period. Spiked Soy Sauce Samples. Dark soy sauce samples confirmed to be free of AFB1 by liquid chromatography coupled with mass spectrometry were purchased from the local market in Nanchang, China. A 10 mL negative sample was fortified by adding AFB1 standard solutions to produce spiked samples containing 0.2, 1.5, and 9 ng/mL of AFB1. The sample extracts were obtained as previously described with some modifications.45 In brief, the dark soy sauce was mixed with ethanol at a ratio of 35:65 (v/v) for 10 min on a vortex shaker, and then the insoluble substance was removed via centrifugation at 10,000 g for 5 min. The supernatants were diluted 6-fold using PBS (0.01 M, pH 7.0) buffer for IMS.


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RESULTS AND DISCUSSION Characterization of MFBs and MFB-mAb Probe. In this study, the MFBs were prepared via an ultrasonic emulsification–solvent evaporation process (Scheme 1A) using a trichloromethane containing OC-QDs, OA-IONPs, PMMA, or PMAO as the oil phase and an SDS aqueous solution as the water phase. The 616 nm emitting OC-QDs with an average size of 8.0 nm and OA-IONPs with an average size of 10.0 nm were used to provide high luminescence and superparamagnetism (Figure S1), respectively. The resultant MFBs in Figure 1A showed a compact IONP-QD-polymer structure with regular spherical shapes and relatively uniform size distribution with an average diameter of 180 ± 20 nm (n = 30). The high-magnification TEM image (Figure 1A) shows that the MFBs possess a quasi-core/shell structure with an obvious shell doped with numerous 8.0–10 nm light and dark dots covered on the core of the polymer matrix. These light and dark dots were inferred to be OC-QDs and OA-IONPs, respectively, because of their different degrees of electron penetrability. The core/shell structure of the MFBs is presumably caused by the hydrophobic difference of OC-QDs, OA-IONPs, PMAO, and PMMA. PMMA is more likely to form a polymer core because of its strong hydrophobicity. Dynamic light scattering analysis showed that the MFBs possess an average hydrodynamic diameter of 180.6 nm with a polymer dispersity index of 0.184, indicating good monodispersity (Figure 1B). The zeta potential of -53.8 mV indicated that the carboxyl groups were successfully modified on the surface of the prepared MFBs. The as-prepared MFBs were expected to possess high luminescence intensity to



provide a high detection sensitivity of ICA and excellent fluorescence stability against changes in external environments to provide high practicability. As shown in Figure 1C, the OC-QDs and MFBs possessed narrow emissions with nearly the same emission peaks at about 616 nm. Moreover, the as-prepared MFBs displayed ca. 226-fold enhanced fluorescence intensities compared with the corresponding QDs (detailed calculation presented in Supporting Information S2). Meanwhile, the fluorescence intensities of the resulting MFBs showed no significant change at the pH range of 3–14 (Figure 1D). Moreover, the average hydrodynamic diameter and fluorescence intensity of the MFBs showed a negligible change after 30 days of storage at room temperature (Figure 1E). These results indicated that the MFBs prepared via ultrasonic emulsification possess a high luminescence with a good colloidal stability and can be used as a fluorescent reporter of ICA. Thereafter, the magnetic property of the MFBs was evaluated using the magnetic property measurement System (SQUID) at 300 K. As shown in Figure 1F, the MFBs and OA-IONPs possessed superparamagnetism with saturation magnetizations of 18.2 and 40 emu/g, respectively. Notably, the saturation magnetization of the MFBs was 45.4% that of the OA-IONPs, which was much higher than the mass percent of IONPs doped in MFBs (11.8%). The high saturation magnetization obtained from a relatively low dosage of IONPs was presumably attributed to the oriented aggregation of IONPs at the outer layer of the MFBs.46 Moreover, the relatively low doping amount of the IONPs helps preserve the fluorescence intensity of the MFBs. As shown in the insert photos (Figure 1F), the MFBs could be readily separated by applying an external


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magnet and possess an extremely bright fluorescence under UV lamp excitation. In conclusion, a simple ultrasonic emulsification process was proposed to prepare bi-functional MFBs, and the resulting sub-micron sized MFBs showed high fluorescence, excellent stability, and good magnetic responsibility. MFB-mAbs were prepared by labeling a saturated content of unpurified anti-AFB1 ascites to MFBs directly via amido bonds, where the mass of miscellaneous proteins in the ascites blocked the excess carboxyl groups of MFBs for decreased nonspecific adsorption of MFB-mAbs on the NC membrane. The saturated content of ascites on the surface of the MFBs was optimized by labeling different amounts of ascites from 25 μg to 600 μg to 1 mg of MFBs. The resultant MFB-mAbs were then applied to run the strip, and the fluorescent signals on the T line (FIT) were used to evaluate the optimal amounts of ascites on the MFBs. Figure S2 shows that the FIT value increased sharply as the ascite amount was increased from 25 μg to 75 μg, and then the FIT values decreased with further increasing ascite content. These results indicate that the steric hindrance of overabundant proteins on the surface of the MFBs reduced the bioactivity of mAbs. Therefore, 75 μg of ascites for 1 mg of MFBs was intended to be the saturated labeled concentration. Compared with that of free MFBs, the average hydrodynamic diameter of MFB-mAbs increased to 213.2 nm (Figure 1B), and the zeta potential remarkably changed from −53.8 mV to -35.1 mV, indicating the successful modification of anti-AFB1 ascites onto MFB surface.



Figure 1. Characterization of the synthesized MFBs. (A) TEM images of the MFBs embedded with OC-QDs and OA-IONPs (insert: individual magnified MFB). (B) Hydrodynamic diameter distribution of MFBs (red line) and MFB-mAbs (blue line). (C) Fluorescence emission spectra of the QDs (blue line) in hexane solution, and the MFBs (red line) in water, respectively. (insert: photographs of MFBs suspension under visible (left) and ultraviolet light (right). (D) Fluorescence intensities of MFBs at different pH values. (E) Fluorescence intensity (red line) and hydrodynamic diameter (blue line) of MFBs dispersed in PBS against the storage time. (F) Magnetic hysteresis loops of MFBs (red line) and OA-IONPs (black line) at 300 K (insert: photographs of MFBs


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retrieved by magnet under visible (left) and ultraviolet light (right).

Optimization of MFB-based ICA. The principle of MFB-based ICA for the sensitive detection of target analytes in complex samples is shown in Scheme 1B. First, MFB-mAbs were added to a large amount of sample solution for specially capturing target analyte. Second, MFB-mAbs and analyte immunocomplexes were collected magnetically and resuspended in a small-volume suspension buffer for running the strip. In the present study, the MFB-ICA for AFB1 detection was developed by dispensing AFB1-BSA conjugates (competing antigen) and donkey anti-mouse IgG on an NC membrane as T and C lines, respectively. To obtain the highest sensitivity and proper signal intensities on both lines, we optimized the concentrations of MFB-mAbs and AFB1-BSA conjugates by using a “checkerboard titration” method,24 which was conducted by serially altering MFB-mAb amounts and AFB1-BSA concentrations at the T area. The concentration of donkey anti-mouse IgG on the C line was designated as 1 mg/mL. The fluorescence intensities on the T and C lines (FIT and FIC) for AFB1-free sample and the competitive inhibition rate for the spiked AFB1 sample (100 pg/mL) were used to verify the optimized parameters. The competitive inhibition rate was calculated by (1−B/B0) × 100%, where B0 and B are FIT/FIC values of AFB1-free and AFB1-spiked solutions, respectively. In theory, low amounts of competing antigen (AFB1-BSA) and MFB-mAbs result in a high inhibition rate (Table S1), whereas insufficient amounts of competing antigen and MFB-mAbs probe cause a low FIT signal, thereby limiting the stability of the proposed ICA. When AFB1-BSA concentration was 0.5 mg/mL and MFB-mAbs



amount was 0.25 μg, the fluorescence signals on the T and C lines were 618 ± 38 and 491 ± 34, respectively, which showed two distinct red bands on both lines under the UV excitation (Figure S4). The competitive inhibition rate for 100 pg/mL AFB1 was achieved at a relatively high value of 55% ± 6%. Thus, the optimal concentration of AFB1-BSA was 0.5 mg/mL sprayed on the test line, and 0.25 μg of MFB-mAbs was used for the running strip. Pretreatment of dark soy sauce for MFB-ICA. Melanoidin in dark soy sauce produces a strong interference to MFB-mAbs and AFB1 interaction even after 1000 times dilution (Figure S3). Therefore, ethanol was added to a final concentration of 65% (v/v) to precipitate the melanoidin in dark soy sauce. However, the high content of ethanol in the pretreated sauce solution could destroy the bioactivity of the MFB-mAbs. To evaluate the effects of ethanol on the capture efficiency of the MFB-mAbs to AFB1, we diluted the pretreated sauce solutions with PBS buffer (pH 7.0) for 2-, 4-, 6-, 8-, and 12-folds and then fortified them with AFB1 stock solution to a final concentration of 100 pg/mL. Then, 0.25 μg of MFB-mAbs was mixed with 70 μL of sauce solutions diluted in series. After incubation at room temperature for 30 min, the retrieved MFB-mAbs by magnetic field were re-suspended with 70 μL of PBS solution containing 5% ethanol for the running strip. The diluted AFB1-free sauce solutions were used as negative experiments. Figure 2A indicates that the maximum FIT value of ca. 600 was achieved in the 4–12-fold diluted sauce solution (without AFB1), whereas the highest inhibition rate was obtained in the 6–12-fold diluted AFB1-spiked sauce sample. These values were almost equal to those in PBS


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buffer, demonstrating that the pretreated sauce solution with 6-fold dilution is enough to eliminate the negative interference of the sauce matrix on MFB-mAbs. Therefore, the pretreated sauce solution was suggested to be further diluted 6-fold with PBS for IMS. Enrichment of AFB1 from a larger volume of sample solution is conducive to improving the sensitivity of ICA. However, the retrieved MFB-mAbs from a large volume would prolong the magnetic separation time, especially for ultralow amounts of MFB-mAbs. In the present study, the amount of MFB-mAbs (0.25 μg) for IMS is only 1/100 of conventional IMS (> 25 µg MBs in 1 mL sample).18,47 Therefore, the separation time to retrieve MFBs was investigated using a series of different volumes of sample solutions from 70 to 700 μL containing equal MFB-mAbs (0.25 μg). The magnetically retrieved MFBs at different separation time were re-suspended in 70 μL PBS buffer for running the strip. As shown in Figure 2B, larger volumes required more separation time to retrieve the MFB-mAbs. Considering a sufficient high enrichment factor and an accepted separation time, 350 μL of sample solution with 20 min of magnetic separation time was selected for succeeding experiments. In addition, the retrieved MFB-mAbs should be dispersed with a suspension solution for the running strip. The pH of the suspension solution could influence the sensitivity of ICA by changing the binding efficiency of MFB-mAbs against antigen on the T line. To explore the effect of pH on immunoreaction between MFB-mAbs and AFB1-BSA at the T zone, 0.01 M PBS buffer with pH 4–9 was applied to run the strip. Figure 2C indicates that the FIT value for the AFB1-free sample increased



sharply from 355 ± 24 to 619 ± 11 as the pH was increased from 4 to 7, retained a relatively constant value at the pH range of 7 to 8, and then significantly decreased as the pH was further increased to 9.0. Higher FIT indicated a higher binding efficiency of MFB-mAbs to AFB1-BSA, thereby improving the detection sensitivity of ICA. The results for the AFB1-spiked sample (100 pg/mL) also showed the same tendency that the competitive inhibition rate increased from 27% to 54% as the pH was increased from 4.0 to 7.0, then declined sharply to 20% as the pH was increased to 9.0. To improve sensitivity, we selected 0.01 M PBS buffer with pH 7.0 as the optimal pH condition for the running strip. Furthermore, a suspension solution containing a certain concentration of ethanol is required for AFB1 detection due to its strong hydrophobicity. Figure 2D indicates that the FIT and FIC values for the AFB1-free sample declined obviously as the ethanol content was increased, and the competitive inhibition rate for the AFB1-spiked sample slightly increased from 58% to 60% as the ethanol content was changed to 5%, whereas the competitive inhibition rate declined sharply to 13% when the ethanol content was further increased to 40%. On the basis of these results, 0.01 M PBS containing 5% (v/v) ethanol with pH 7.0 was used to resuspend the retrieved MFB-mAbs for the running strip.


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Figure 2. Optimization of the MFB-ICA Sensor. (A) Effect of dilution rates of pretreated soy sauce on FIT and inhibition rate. Red dotted line is the inhibition rate for 100 pg/mL AFB1 in 70 μL PBS buffer (0.01 M, pH 7.0). Blue dotted line is the FIT for 0.25 μg MFB-mAbs in 70 μL blank PBS buffer (0.01 M, pH 7.0). (B) Effect of magnetic separation time of samples on FIT in different reaction system. (C) Effect of pH value of resuspension buffer on competitive inhibition rate of an AFB1 spiked sample (100 pg/mL). (D) Effect of ethanol in resuspension buffer on competitive inhibition rate. The data derived from the mean ± SD of three separate experiments.

Analytical performance of MFB-ICA for AFB1 detection in dark soy sauce. In this study, the FIT/FIC ratio was used for AFB1 quantitative assay because it can effectively offset the inherent heterogeneity of strips, and immunological kinetics analysis was performed to optimize the interpretation time of the MFB-ICA for AFB1 quantitative analysis according to our previous works.24 It was performed by running a series of samples containing certain amounts of AFB1 (0, 20, 50, and 200 pg/mL, respectively) on strips. After running for 1 min, the FIs on both lines and FIT/FIC ratio



were recorded every 1 min for 35 min. The results in the insert of Figure 3A showed that the FIs on the T and C lines enhanced consecutively within 35 min, whereas the FIT/FIC value achieved a balance after 12 min under the different AFB1 concentrations. To ensure the accuracy of the MFB-ICA, a 15 min of running strip time was employed for AFB1 quantitative analysis. Under the developed conditions, the calibration curve of the MFB-ICA for AFB1 quantitative detection was described by plotting the B/B0 × 100 vs. the logarithm of AFB1 concentrations (0–500 pg/mL). The AFB1 standard solutions were obtained by spiking a stock solution (1.0 µg/mL) to the AFB1 free dark soy sauce extract, which was prepared via a 6-fold dilution of the ethanol-pretreated dark soy sauce with PBS buffer. The result in Figure 3B shows that the calibration curve exhibits a good linear range for AFB1 quantitative detection from 5 pg/mL to 150 pg/mL (Figure S4). The regression equation was represented by y = -17.69 ln(x) + 108.49 (R2 = 0.9931), where x and y represent AFB1 concentrations and the competitive inhibition rate. The IC50 value of the MFB-ICA was achieved at 27 ± 3 pg/mL (n = 3), whereas the LOD was 3 pg/mL. The LOD is defined as the corresponding AFB1 concentration that produces 10% competitive inhibition rate by the MFB-ICA. The LOD of the proposed method for a real dark soy sauce was calculated to be 49 pg/mL by the LOD of soy sauce extract multiplied with the dilution factor (DF). The DF was calculated to be 17.1 by using the following formula: DF= Vsum/Vsample, where Vsum is the final volume after ethanol extraction and successive dilution, and Vsample is the volume of the initial soy sauce sample. Currently, several available fluorescence-based ICA methods have


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been presented for AFB1 detection in soy sauce. For example, Liu et al. developed a FITC-doped polystyrene NP-based ICA for AFB1 detection in soy sauce with a LOD of 2.5 μg/L.48 In addition, Zhang et al. and Wang et al. respectively used Eu(III)-marked polystyrene beads49 and Eu(III)-doped nanospheres50 as an amplification label of ICA for AFB1 in soy sauce, and the LODs were as low as 0.09 μg/kg and 0.1 μg/kg. Recently, Jiang et al. reported fluorescence-quenching ICA based on the inner filter effect for AFB1 ultrasensitive detection.51 The LOD value was reached at 40 pg/mL in light soy sauce. In a word, the LOD of the proposed MFB-ICA method is far superior to those of previously reported fluorescence-based ICA methods, and is comparable to that of reported fluorescence-quenching ICA for AFB1 detection in soy sauce (Table S2).

Figure 3. (A) Immunoreaction dynamics monitoring of FIT, FIC and FIT/FIC against AFB1 concentration and time. (B) Standard inhibition curve for AFB1 obtained by using 350 μL pretreated soy sauce. (C) Cross-reactivity of MFB-ICA to nonspecific toxins. Samples are AFB1



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(200 pg/mL), AFG1 (200 pg/mL), AFB2 (10 ng/mL), FB1 (100 ng/mL), OTA (100 ng/mL), DON (100 ng/mL), ZEN (100 ng/mL), CIT (100 ng/mL), a negative control (0.01 M PB, pH 7.0, 5% ethanol). The data derived from the mean ± SD of five separate experiments.

The selectivity of the MFB-ICA sensor was estimated through analyzing two common structural analogues, namely, AFG1 (200 pg/mL) and AFB2 (10 ng/mL), as well as five other common mycotoxins (100 ng/mL), namely, fumonisin, ochratoxin A, deoxynivalenol, zearalenone, and citrinin. Figure 3C indicates that the developed MFB-ICA exhibits a certain extent of cross-reactivity (CR) with AFG1, and negligible CR with other six mycotoxins. Then, the accuracy and precision of our MFB-ICA sensor were evaluated by performing the recovery studies of intra- and inter-assay with the spiked sauce extracts under three different AFB1 contamination levels of low (0.2 ng/mL), medium (1.5 ng/mL), and high (9.0 ng/mL), respectively. The detection details for AFB1 intra- and inter-assay could be found in our previous publication.24 Results in Table 1 exhibit that the average recoveries for the intra-assay changed from 89% to 103%, with a coefficient of variation (CV) ranged from 1% to 5%, whereas the average recoveries for the inter-assay changed from 93% to 107% with a CV from 3% to 7%, respectively, revealing an acceptable accuracy and precision for AFB1 quantification via using the MFB-ICA Table 1. The accuracy and precision of the MFB-ICA method in AFB1-spiked dark soy sauce Inter-assaya

Intra-assay

Spiked AFB1 (ng/mL)

Meanb

Recovery (%)

CV (%)

Meanb

Recovery (%)

CV (%)

0.2

0.2 ± 0.0

97

1

0.2 ± 0.0

96

3

1.5

1.5 ± 0.1

103

5

1.6 ± 0.1

107

7

9.0

8.0 ± 0.2

89

3

8.4 ± 0. 6

93

7

aAssay bMean

was completed every 1 days for 3 days continuously. value of five replicates at each spiked concentration.


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The robustness of the proposed method was evaluated by analyzing six real dark soy sauce samples that were randomly and artificially polluted by AFB1. These samples were further confirmed by ultra-performance liquid chromatography with fluorescence detection in accordance with the national standard GB 5009.22-2016 of China (details shown in supporting information S8). Table 2 showed that 4 of 6 samples were tested with AFB1 pollution using these two methods, and 2 samples were detected without AFB1 by both methods, indicating a good agreement between these two methods. These results indicate that the proposed method exhibits great potential for trace pollutant analysis in complex matrix samples. Table 2. Analysis of AFB1 contamination in real dark soy sauce samples by UPLC and MFB-ICA methods

a Mean

Soy materials

MFB-ICA (ng/mL)a

UPLC (ng/mL)

dark soy sauce no.1 dark soy sauce no.2 dark soy sauce no.3 dark soy sauce no.4 dark soy sauce no.5

－b － 15.0 ± 0.2 1.7 ± 0.3 153 ± 18

－－ 16.1 1.5 138

dark soy sauce no.6

1.7 ± 0.1

1.7

of three repeated determinations. b Not detected.



CONCLUSIONS We prepared bifunctional MFBs with a core/shell structure via a facile one-pot ultrasonic emulsification. The resultant MFBs displayed ca. 226-fold stronger fluorescence emission relative to the corresponding QDs and retained ca. 45.4% of the saturation magnetization compared with the OA-IONPs. Compared with conventional magnetic or fluorescent materials, MFBs exhibited more advantages as an ICA reporter for trace AFB1 detection from complex food matrix, such as dark soy sauce. Compared with the conventional fluorescence-based ICA, the MFB-ICA showed a high sensitivity and good reproducibility via the magnetic enrichment of target analytes and the elimination of matrix interference. This result was verified by the determination of AFB1-spiked dark soy sauces. In addition, the developed MFB-ICA showed a good agreement with the UPLC-FLD method for analyzing AFB1 in real dark soy sauce samples. In brief, the proposed MFB-ICA provides a versatile and easy strategy to improve the analytical performance in rapid screening mycotoxins or other pollutants in complex food or biological samples. In addition, the MFB-ICA can achieve multi-signal readout by fluorescence or magnetic signal for quantitative detection and by colorimetric signal for visual detection.


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ASSOCIATED CONTENT Supporting Information TEM images of OC-QDs and OA-IONPs (Figure S1); Comparison of fluorescence intensity between CdSe/ZnS QDs and the as-prepared MFBs; Confirmation of the saturation concentration of the mAb conjugated with the MFBs (Figure S2); Optimization of the QBs strip parameters (Table S1); The strong interference of the melanoidin in dark soy sauce (Figure S3); The typical strip tests of standard solutions (Figure S4); Comparison of reported fluorescent signal based ICA with MFB-ICA for AFB1

detection

in

soybean

sauce

(Table

S2);

Ultra-performance

liquid

chromatography (UPLC) analysis. ACKNOWLEDGMENTS This research was supported by the National Key Research and Development Program of China (2018YFC1602203, 2018YFC1602505, 2018YFC1602202,), the National Natural Science Foundation of China (No. 31360385) and the Science & Technology Transformation Program of Jiangxi Province (No. KJLD13011). NOTES The authors declare no competing financial interests. REFERENCES (1) Almeida, M. I. G. S.; Jayawardane, B. M.; Kolev, S. D.; McKelvie, I. D. Talanta 2018, 177, 176-190.



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