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.
Silk fibroin (SF) has been engineered in the biomedical applications on account of its structural robustness, biocompatibility, and biodegradability. However, in situ study is still lacking with respect to the formation of SF secondary structures at the interface. In this paper, by using methanol as an inducing agent, the formation of SF secondary structures at the polystyrene (PS)/SF solution interfaces was detected with achiral and chiral sum frequency generation (SFG) vibrational spectroscopy. SF solutions with two concentrations above and below the critical overlapping concentration ( C*) of SF (∼1.8 mg/mL) were chosen, namely, 90 and 1 mg/mL. We found that above C*, before adding methanol to the protein solution, no ordered SF secondary structures could be detected at the PS/SF solution interface; oppositely, after adding methanol to the protein solution, ordered SF secondary structure, for example, antiparallel β-sheet, could be formed at the PS/protein solution interface. Below C*, both before and after adding methanol to the SF solution, ordered SF secondary structure such as antiparallel β-sheet could be formed. Besides, the addition of methanol could induce the formation of an extended helical structure, verified by the achiral and chiral characteristic bands. Because C* represents a critical solution concentration above which the SF chains can interact with each other and below which the SF chains are isolated in the solution, this achiral/chiral SFG study emphasizes the importance of the chain-chain interaction or spatial confinement on the formation of the protein secondary structures, which provides an additional dimension for the future study of interfacial protein folding.
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