Development and Application of a Quantitative Fluorescence-Based

Jun 14, 2014 - A fluorescence-based immunochromatographic assay (ICA) for fumonisin B1 (FB1) that employs conjugates of fluorescent microspheres and m...
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Development and application of a quantitative fluorescencebased immunochromatographic assay for fumonisin B1 in maize Zhanhui Wang, Heng Li, Chenglong Li, Qing Yu, Jianzhong Shen, and Sarah De Saeger J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 14 Jun 2014 Downloaded from http://pubs.acs.org on June 21, 2014

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Development and Application of a Quantitative

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Fluorescence-based Immunochromatographic Assay

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for Fumonisin B1 in Maize

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Zhanhui Wang,† Heng Li,† Chenglong Li,† Qing Yu,† Jianzhong Shen,*,† Sarah De

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Saeger§

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for Food Quality and Safety, Beijing Key Laboratory of Detection Technology for

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Animal-Derived Food Safety , Beijing 100193, People’s Republic of China

College of Veterinary Medicine, China Agricultural University, Beijing Laboratory

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§

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Harelbekestraat 72, 9000 Ghent, Belgium

Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University,

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* Author to whom correspondence should be addressed

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Tel: +86-10-6273 3289

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Fax: +86-10-6273 1032

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E-mail: [email protected]

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ABSTRACT

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A fluorescence-based immunochromatographic assay (ICA) for fumonisin B1

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(FB1) that employs conjugates of fluorescent microspheres and monoclonal antibodies

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(FM-mAbs) as detection reporters is described. The ICA is based on the competitive

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reaction between FB1-BSA (BSA, bovine serum albumin; test line) and the target FB1

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for binding to the FM-mAb conjugates. A limit of detection (LOD) for FB1 of 0.12

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ng/mL was obtained, with an analytical working range of 0.25-2.0 ng/mL

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(corresponding to 250-2000 µg/kg in maize flour samples, according to the extraction

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procedure). The recoveries of the ICA to detect FB1 in maize samples ranged from

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91.4% to 118.2%. A quantitative comparison of the fluorescence-based ICA and

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HPLC-MS/MS analysis of naturally contaminated maize samples indicated good

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agreement between the two methods (r2=0.93). By replacing the target of interest, the

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FM-based ICA can easily be extended to other chemical contaminants and thus

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represents a versatile strategy for food safety analysis.

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KEYWORDS: fumonisin B1, fluorescent microsphere, immunochromatographic

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assay, maize, mycotoxins

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 INTRODUCTION

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Rapid assays for the detection of a variety of mycotoxins in food or feed are

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generating ever-increasing scientific and technological interest because these assays

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enable simple, one-step, in situ analyses.1,2 Among these rapid assays, enzyme-linked

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immunosorbent assays (ELISAs) and immunochromatographic assays (ICAs) are two

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well-established and accepted techniques.3-5 In practical applications, ICAs, which

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typically use colloidal gold as the reporter, have primarily been used for the

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qualitative detection of mycotoxins that are frequently present at relatively high

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concentrations.6-8 Applications of ICAs have been limited by low sensitivity and poor

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quantitative discrimination, which are intrinsically determined by the molar

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absorption coefficient of the gold nanoparticles and the accumulating ability of the

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analyte, respectively.9 The analysis of mycotoxins with increased sensitivity and

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accuracy requires reporter systems that better enable high analytical sensitivity and

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quantitative detection. Recently, a variety of reporters, including fluorescent dyes,

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liposomes,

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superparamagnetic nanoparticles, and fluorescent europium nanoparticles, have been

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developed and used in an ICA format for the detection of different targets of

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interest.10-15 However, less marked improvement in performance has been achieved,

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and alternative reporters remain to be developed.

quantum

dots,

and

particles

such

as

silica

nanoparticles,

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Fluorescent microspheres (FMs) are polystyrene materials that contain dyes in

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the interior of the bead instead of merely on the surface of the bead, thereby

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producing a stable configuration, high fluorescence intensity, and photostability.16,17

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They also exhibit a narrow distribution of fluorescence intensities and sphere sizes

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and are available in an array of colors; thus, they are potentially more accurate and

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diverse than currently used reporters. FMs are frequently employed in suspension

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array technologies as encoding probes, but their use as labels in the ICA format has

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not been reported.18-20

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Contamination of cereals and related product by mycotoxins has become an

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increasingly serious problem. Consumers are concerned by public health-related

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issues and show high preoccupation about the risks associated with human exposure

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to mycotoxins.2 Fumonisins (FBs) are nephrotoxic, hepatotoxic and carcinogenic

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mycotoxins mainly produced by Fusarium mould species (primarily F. verticillioides

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and F. proliferatum). There are many different forms of FBs; among these, FB1

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usually constitute about 70% of the total FBs content found in naturally contaminated

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foods and feeds.4,7 Due to its toxic to animal and human, the FB1 has been declared by

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the International Agency for Research on Cancer (WHO) to be a group 2B

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carcinogen.21

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In this work, we describe the development of a novel FM-based ICA for the

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quantitative detection of FB1 as a model mycotoxin in maize samples. FMs were used

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to label anti-FB1 monoclonal antibodies (mAbs) to improve the sensitivity of the

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assay. The analytical performance of the ICA was validated by determining the levels

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of FB1 in maize samples using an HPLC-MS/MS method as a reference method.

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 MATERIALS AND METHODS 4

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Reagents. FB1 (purity ≥95.0%), FB2 (≥97.0%), and FB3 (≥97.0%) were

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purchased from Fermentek, Ltd. (Jerusalem, Israel). Zearalenone, ochratoxin A,

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deoxynivalenol, aflatoxin B1 and T-2 toxin were purchased from Acros Organics Co.

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(Worcester, MA). Fluospheres carboxylate-modified microspheres, red fluorescent

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(580/605, λex/λem), 2% solids, were purchased from Invitrogen (Carlsbad, CA). Bovine

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serum albumin (BSA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

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hydrochloride (EDC), and 2-(N-morpholino) ethanesulfonic acid (MES) were

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purchased from Sigma-Aldrich (St. Louis, MO). All other reagents were analytical

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grade or better and were purchased from Beijing Reagent Corp. (Beijing, P.R. China).

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The nitrocellulose filter membrane (HF13520s25) was obtained from Millipore

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Corporation (Bedford, MA). The sample pad (CH37K) and the absorbance pad (SB08)

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were supplied by Shanghai Liangxin Co., Ltd. (Shanghai, China). White opaque

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96-well polystyrene microtiter plates were purchased from Costar, Inc. (Milpitas, CA).

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The mAb 2D7 to FB1 and the coating antigen were prepared previously and were

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purified using Protein A prior to use.22

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MES buffer was prepared as follows: 9.76 g of MES was dissolved in 1000 mL of purified water. The pH values of MES buffer were adjusted by 1M NaOH. Apparatus. The NanoDrop ND-1000 spectrophotometer was purchased from

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Gene Company, Ltd. (Hong Kong, P.R. China). The ZX1000 dispensing platform and

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the CM4000 guillotine cutting module used to prepare the ICA were purchased from

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BioDot, Inc. (Irvine, CA). The ESE Quant LFR fluorescence reader was purchased

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from QIAGEN (Dusseldorf, Germany). The UV spectrometer was provided by

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Qiangyun Co. (Shanghai, P.R. China).

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Preparation of FM-labeled FB1 mAbs. The FM-mAb conjugates were

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prepared as follows: anti-FB1 mAb (7 µg) was dissolved in 1 mL of 50 mM MES (pH

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6.5), and FMs (10 µL) were added dropwise. After 15 min, EDC (0.5 mg) was added

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and maintained on a shaker for 2 h at room temperature. The pH of the reaction

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mixture was adjusted to 6.5 using 1 M NaHCO3. To quench the reaction, 100 µL of

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0.1 M glycine was added and incubated for 30 min. The FM-labeled mAbs were

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obtained after centrifugation at 8,076 g for 15 min at room temperature to remove any

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unreacted mAbs. The pellets were resuspended in 100 µL of 0.05 M PBS containing

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0.2% BSA and 0.4% polyethylene glycol. The suspension was dispersed and blended

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by supersonic vibration for 10 min and stored at 4 °C in the dark until use.

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Fluorescence-based ICA. Suspensions of 0.1 mg/mL FB1-BSA and 1 mg/mL

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goat anti-mouse IgG were dispensed onto nitrocellulose filter membranes to produce

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the test (0.7 µL/cm) and control (0.9 µL/cm) lines, respectively. The membrane was

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dried for 2 h at 37 °C in an air oven. The sample pad was soaked in 0.01 M PBS

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(containing 0.05% Tween 20 and 0.05% sodium azide) and dried at 40 °C for 24 h.

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The absorbent pad was pasted onto the top side of the backing pad by overlapping a

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2-mm section with the membrane. The sample pad was also pasted onto the other side

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of the backing pad by overlapping a 2-mm section with the membrane. Finally, the

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whole assembled plate was cut into 4-mm-wide strips and stored under dry conditions

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at room temperature. A total of 120 µL of sample solution (or standard buffer) was

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transferred into a 96-well microtiter plate and homogenized with 2 µL of FM-mAbs at

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room temperature. After 5 min of shaking, the strip was added to the microwell to

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absorb the mixture. The capillary migration process lasted approximately 15 min, and

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the strip was then removed and fully dried for 5 min at 40 °C in an air oven prior to

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measuring the fluorescence signal. The results of the test and control lines were either

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qualitatively estimated by eye under a UV light or quantitatively measured by

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measuring the signal intensities using an ESE Quant lateral flow reader set at 580 and

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605 nm for the excitation and emission wavelengths, respectively.

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To test the specificity of the ICA for FB2, A,

zearalenone,

T-2

and aflatoxin

toxin, deoxynivalenol

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(DON),

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of these mycotoxins with concentration of 0.1, 0.5, 1, 1.5, 2.0, 4.0 and 10 ng/mL were

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prepared by diluting the corresponding stock solutions (1 mg/mL) with 0.01 M PBS.

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The following ICA experiments were carried out in the same way as mentioned above

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for FB1. The cross-reactivity values were calculated according to the following

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equation:

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ochratoxin

FB3,

B1. The standard solution

[IC50 (FB1, ng/mL)/IC50 (mycotoxins, ng/mL)] × 100%

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Analysis of Maize Samples. Maize flour samples were provided by local

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company in Belgium and stored at -20 °C. The concentration of mycotoxins was

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determined by HPLC-MS/MS, as previously described.23 In spike and recovery

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studies, 10 g of FB1-negative maize flour samples were spiked with FB1, which was

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dissolved in methanol, with 500, 1000, and 1500 µg/kg. The samples were thoroughly

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mixed and then allowed to stand at room-temperature overnight. Maize flour (1 g) 7

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was vigorously mixed and extracted with 20 mL of methanol/water (4:6, v/v) for 5

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min before ultrasonic dispersion for 5 min and centrifugation at 8,076 g for 5 min. A

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total of 200 µL of supernatant was diluted with 800 µL of sample diluent prior to

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analysis by the ICA as described before.

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 RESULTS AND DISCUSSION

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Detection Principle of the FM-based ICA. In this study, the detector reagent

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consisted of anti-FB1 mAb-functionalized FMs. The carboxyl groups of fluorescent

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microsphere were covalently coupled to the amino group of mAb in the presence of

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carbodiimide to insure the stability of the detector (Figure 1). The detection principle

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of the FM-based ICA was based on the competitive binding between the FB1-BSA

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(Test line) and the mycotoxin FB1 to combine with the limited mAbs on the

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fluorescence reporter. As shown in Figure 1, when FB1 was present in the sample

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solution, the FB1-FM-mAbs were formed in a microwell, diminishing the formation

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of FB1-BSA-FM-mAbs at the test line causing its fluorescence intensity to become

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weaker. Regardless of the presence of FB1 in the sample, secondary antibody in the

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control line would bind to the anti-FB1 mAbs ensuring the validity of the detection.

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According to the principle described above, the fluorescence intensity on the test line

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would be inversely proportional to FB1 concentrations in the sample, which could be

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used for quantitation of FB1 in samples. Quantitative analysis was realized by reading

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the fluorescence intensities of test line with a portable strip reader.

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FM-labeled mAbs. The activity of the FM-mAbs primarily guaranteed assay

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speed and sensitivity; thus, we first optimized the FM-mAb preparation conditions.

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Figure 2A shows the influence of the FM diameter on the fluorescence intensity of the

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test line. In the presence of 300-nm FMs, the signal generation was rather slow, and it

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took more than 30 min to accomplish the procedure. Although the 300-nm FMs

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produced strong signals, this duration is not appropriate for a rapid assay (Figure 2A).

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Besides, the significant residue of FM-mAb conjugates in the membrane pores could

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affect the accurate detection of the target (Figure 2A). It took less than 10 min to

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accomplish the procedure in the presence of 100-nm FMs; however, the signal

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response was very weak, as shown in Figure 2A, indicating that the small-diameter

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FMs were not appropriate for application with the ICA. The 200-nm FMs represented

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a compromise between speed and sensitivity in the assay and were selected for further

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experiments (Figure 2A).

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The pH of the buffer has a great influence on the fluorescence of the FMs alone

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and on the FM-mAb conjugates, e.g. through their dissolution and stability properties;

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therefore, the effect of several buffers with different pH values on these FM properties

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was evaluated. The fluorescence signal of FMs alone has been tested at pH values

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outside 5.0-6.5, and the signal of FM-mAb conjugates at pH values between 5.0 and

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6.5 (Figure 2B). Thus, we evaluated the effect of buffers with pH values of 5.0, 6.0

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and 6.5 on the performance of the FM-mAb conjugates. The FM-mAb conjugates

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clogged the membrane at pH values of 5.0 and 6.0. The pH 6.5 coupling buffer was

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thus selected as the most suitable condition for the subsequent experiments.

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Figure 2C shows the results of the mAb loading onto the FMs. The fluorescence

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intensity of the FMs increased as increasing amounts of mAb were added onto the

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FMs until asymptotically approaching a maximum value. As the increase of intensity

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is very limited for amounts of added mAb above 7 µg (Figure 2C), this quantity has

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been selected as the most appropriate for an adequate signal sensitivity in the ICA.

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One of the main characteristics of ICA is rapid detection, which is an important

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advantage over other immunoassays. A time-intensity curve was constructed by

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measuring the signal of the test line in function of incubation time. The signal

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increased rapidly during the first 15 min, and the signal asymptotically reached a

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maximum for longer incubation time plateaued between 15 and 30 min (Figure 2D),

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(Figure 2D), indicating that the signal was already relatively stable for analysis at

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15-30 min. With consideration of finding the shortest time for an optimal detection,

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the best incubation time was considered as 15 min.

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Analytical Performance of the ICA. The fluorescence ICA is based on

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competitive binding of the FB1 in the samples and the FB1-BSA immobilized on the

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test line to the FM-mAb conjugates flowing through the membrane. Analytical

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parameters of the ICA for FB1 were obtained under the previously determined optimal

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conditions. Concentrations of FB1 from 0-3.0 ng/mL were applied to the ICA, and the

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corresponding inhibition fluorescence intensity of the test line was observed under an

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ultraviolet light source or recorded by a fluorescence reader. As shown in Figure 3A,

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the red fluorescence intensity of the test line clearly decreased as the concentration of

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FB1 increased. The fluorescence intensity was barely detectable at 2.5 ng/mL of FB1;

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thus, 2.5 ng/mL of FB1 was considered the cutoff value for the assay (corresponding

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to 25 µg/kg in maize flour samples according to the extraction procedure used in the

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study). A calibration curve was generated using FB1 in the range of 0.25 to 2.0 ng/mL

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(250-2000 µg/kg in maize flour samples) with an IC50 values of 1.32 ng/mL, as shown

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in Figure 3B (r2 = 0.9949). The LOD for the quantitative detection was estimated

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from the concentration that corresponded to the blank test line value minus 3 times the

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standard deviation of the blank, which was calculated as 0.12 ng/mL (1.2 µg/kg in

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maize flour samples). This LOD is lower than that of the ELISA method employing

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the same immunoreagents (5.4 µg/kg in maize flour samples).22

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The specificity of the ICA was determined by evaluating cross-reactivity with

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frequently occurring mycotoxins, including FB2, FB3, T-2 toxin, deoxynivalenol

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(DON), ochratoxin A, zearalenone, and aflatoxin B1. The ICA exhibited

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cross-reactivities with FB2 and FB3 of 1.5% and 67.3%, respectively, and negligible

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cross-reactivities with other mycotoxins (