Development and Application of a Quantitative Fluorescence-Based

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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

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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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†

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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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ACS Paragon Plus Environment


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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 (

Development and Application of a Quantitative Fluorescence-Based

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