The detecting labels used for lateral flow immunoassays (LFAs) have been traditionally gold nanoparticles (GNPs) and, more recently, luminescent nanoparticles, such as quantum dots (QDs). However, these labels have low sensitivity and are costly, in particular, for trace detection of mycotoxins in cereals. Here, we provided a simple preparation procedure for amorphous carbon nanoparticles (ACNPs) and described multiplex LFAs employing ACNPs as labels (ACNP-LFAs) for detecting three Fusarium mycotoxins. The analytical performance of ACNPs in LFA was compared to GNPs and QDs using the same immunoreagents, except for the labels, allowing for their analytical characteristics to be objectively compared. The visual limit of detection for ACNP-LFAs in buffer was 8-fold better than GNPs and 2-fold better than QDs. Under optimized conditions, the quantitative limit of detection of ACNP-LFAs in maize was as low as 20 μg/kg for deoxynivalenol, 13 μg/kg for T-2 toxin, and 1 μg/kg for zearalenone. These measurements were much lower than the action level of these mycotoxins in maize. The accuracy and precision of the ACNP-LFAs were evaluated by analysis of spiked and incurred maize samples with recoveries of 84.6-109% and coefficients of variation below 13%. The results of ACNP-LFAs using naturally incurred maize samples showed good agreement with results from high-performance liquid chromatography-tandem mass spectrometry, indicating that ACNPs were more sensitive labels than and a promising alternative to GNPs used in LFAs for detecting mycotoxins in cereals.

To develop a sensitive fluorescence polarization immunoassay (FPIA) for screening the zearalenone class of mycotoxins in maize, two new monoclonal antibodies with uniform affinity to the zearalenone class and four fluorescein-labeled tracers were prepared. After careful selection of appropriate tracer-antibody pairs in terms of sensitivity and specificity, a FPIA that could simultaneously detect the zearalenone class with similar sensitivity was developed. Under optimum conditions, the half maximal inhibitory concentrations of the FPIA in buffer were 1.89, 1.97, 2.43, 1.99, 2.27, and 2.44 μg/L for zearalenone, α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol, and zearalanone, respectively. The limit of detection of FPIA for the zearalenone class was around 12 μg/kg in maize, and the recoveries ranged from 84.6 to 113.8%, with coefficients of variation below 15.3% in spiked samples. Finally, the FPIA was applied for screening naturally contaminated maize samples, and the results indicated a good correlation with that of high-performance liquid chromatography-tandem mass spectrometry.

Molecular recognition between a receptor and ligand is a fundamental event in bioanalytical assays, which guarantees the sensitivity and specificity of an assay for the detection of the target of interest. An intensive understanding of the interaction mechanism could be useful for desirable hapten design, directed antibody evolution in vitro, and assay improvement. To illustrate the structural information on classspecific monoclonal antibodies (mAbs) and dihydropteroate synthase (DHPS) against sulfonamides (SAs) we have previously prepared, we initially measured the kinetic parameters of mAb 4C7, 4D11, and DHPS, which showed that the affinities of 4C7 and 4D11 were in the pM to μM range, while DHPS was uniformly in the μM range. Threedimensional quantitative structure−activity relationship analysis for 4C7 and 4D11 then revealed that the contributions from the stereochemical structure and electron density of the SAs were comparable to binding with mAb. To acquire insights into the structural basis of mAbs and DHPS during the recognition process, the crystal structures of 4C7 and its complex with sulfathiazole were determined using X-ray crystallography. The results showed the SAs orientation and hydrogen bonding interactions mainly contributed to the diverse SAs-mAb affinities. However, for DHPS, a nucleophilic substitution reaction occurred during the recognition process with the SAs, which contributed to the surprisingly uniform affinity for all the SAs tested. This study verified the previous hypotheses on antibody production against SAs and enhanced our understanding of antibody−SAs interactions, which provided useful information toward the rational design of novel haptens and directed evolution to produce class-specific antibodies as DHPS.

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