A novel, sensitive, and high throughput competitive immunoassay for multiplex mycotoxins was established by immobilizing the artificial antigens (Ags) of mycotoxins on the surfaces of three kinds of silica photonic crystal microsphere (SPCM) suspension arrays. The SPCMs were encoded by their reflectance peak positions. Aflatoxin B1 (AFB1), fumonisin B1 (FB1), and citrinin (CIT) spiked in the cereals were extracted, and the fluorescein isothiocyanate (FITC) labeled antibodies (Abs) of these mycotoxins were added into the centrifuge tube which contained the SPCMs of the modified artificial antigens (Ags). The fluorescence signal was collected by an array fluorescent scanner. The limit of detection (LOD) was as low as 0.5, 1, and 0.8 pg/mL for AFB1, FB1, and CIT, respectively. The new method provided a wide linear detection range from 0.001 to 10, 0.001 to 10, and 0.001 to 1 ng/mL for AFB1, FB1, and CIT, respectively. The mean recovery rates are in range of 74.7 ± 4.0% to 127.9 ± 4.4% for the three mycotoxins in corn, peanuts, and wheat. The developed method for mycotoxins was used to assay the AFB1, FB1, and CIT level in 10 naturally contaminated cereal samples, and the results of detection were in agreement with that of a classic enzyme-linked immunosorbent assay (ELISA) method. This method saves a large amount of reagents (10 μL volume) and detection time (<3 h) for multiplex mycotoxin assay.

A new aptamer microarray method on the TiO-porous silicon (PSi) surface was developed to simultaneously screen multiplex mycotoxins. The TiO nanolayer on the surface of PSi can enhance the fluorescence intensity 14 times than that of the thermally oxidized PSi. The aptamer fluorescence signal recovery principle was performed on the TiO-PSi surface by hybridization duplex strand DNA from the mycotoxin aptamer and antiaptamer, respectively, labeled with fluorescence dye and quencher. The aptamer microarray can simultaneously screen for multiplex mycotoxins with a dynamic linear detection range of 0.1-10 ng/mL for ochratoxin A (OTA), 0.01-10 ng/mL for aflatoxins B (AFB), and 0.001-10 ng/mL for fumonisin B (FB) and limits of detection of 15.4, 1.48, and 0.21 pg/mL for OTA, AFB, and FB, respectively. The newly developed method shows good specificity and recovery rates. This method can provide a simple, sensitive, and cost-efficient platform for simultaneous screening of multiplex mycotoxins and can be easily expanded to the other aptamer-based protocol.

A simple, new aptamer-photonic crystal encoded suspension array was designed to simultaneously quantify and qualify ochratoxin A(OTA) and fumonisin B1(FB1) in cereal samples. The aptamers of OTA and FB1 were immobilized on the surfaces of photonic crystals by chemical bonding. When the target mycotoxins appear in a sample, the fluorescence-labeled complementary DNA of the aptamer dissociates from their double DNA hybrid and results in an obvious decrease in fluorescence intensity of the microsphere. The difference value of fluorescent intensities for each kind of silica photonic crystal microsphere (SPCM) quantitatively conveys the concentration of mycotoxin, and the structure colors or reflectance peak positions of the SPCMs confirm the kind of mycotoxin detected. The reaction conditions including the immobilization method for aptamers, hybridization, and incubation conditions have been optimized. This developed method displayed a wide linear detection range (0.01-1 ng/mL for OTA and 0.001-1 ng/mL for FB1) and a low limit of detection (0.25 pg/mL for OTA and 0.16 pg/mL for FB1). The recovery rates in the spiked cereal samples ranged from 81.80% to 116.38% for OTA and 76.58%-114.79% for FB1. The positive detection results in the naturally contaminated cereal samples were in agreement with those of classic enzyme-linked immunosorbent assay (ELISA). This simple suspension array scheme displays a great application potential for the high throughput screen assay of mycotoxins.

A novel multiplex chemiluminescent mycotoxin immunoassay suspension array system was developed by combining the silica photonic crystal microspheres (SPCMs) encoding technique and a chemiluminescent immunoassay (CLIA) method. The SPCMs were used as a carrier of the suspension array and encoded by their reflectance peak positions, which overcome fluorescence photobleaching, and the potential interference between the encoding fluorescence and detection fluorescence. Aflatoxin B1 (AFB1), fumonisin B1 (FB1) and ochratoxin A (OTA) artificial antigens were immobilized on the surfaces of SPCMs by using 3-glycidoxypropyltrimethoxysilane as a linker. Horseradish peroxidase (HRP) was used as a labeling enzyme for the secondary antibody in the enzyme-catalyze H2O2-luminol chemiluminescence system. The CLIA detection system was easily integrated with a multifunctional microplate reader and displayed a two to three orders of magnitude dynamic linear detection range from 0.001 to 1, 0.001 to 1, and 0.01 to 1 ng mL(-1) for AFB1, FB1 and OTA with 50% inhibitory concentrations (IC50) of 0.01, 0.036, and 0.04 ng mL(-1), respectively. The recovery rates are in the range of 63.5 to 121.6% for the three mycotoxins in three kinds of spiked cereal samples. The results of detection in 12 naturally contaminated cereal samples were consistent with that of the classic enzyme-linked immunosorbent assay (ELISA) method. This proposed system is simple, rapid, low cost and high throughput for multiplex mycotoxin assay.

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