"Lollipop" particle counting immunoassay based on antigen-powered CRISPR-Cas12a dual signal amplification for the sensitive detection of deoxynivalenol in the environment and food samples.

Deoxynivalenol is one of the most widely distributed mycotoxins in cereals and poses tremendous threats to the agricultural environment and public health. Therefore, it is particularly important to develop sensitive and interference-resistant deoxynivalenol analysis methods. Here, we establish a "Lollipop" particle counting immunoassay (LPCI) based on antigen-powered CRISPR-Cas12a dual signal amplification. LPCI achieves high sensitivity and accuracy through antigen-powered CRISPR-Cas dual signal amplification combined with particle counting immunoassay. This strategy not only broadens the applicability of the CRISPR-Cas system in the field of non-nucleic acid target detection; it also improves the sensitivity of particle counting immunoassay. The introduction of a polystyrene "lollipop" immunoassay carrier further enables efficiently simultaneous pre-treatment of multiple samples and overcomes complex matrix interference in real samples. The linear detection range of LPCI for deoxynivalenol was 0.1-500 ng/mL with a detection limit of 0.061 ng/mL. The platform greatly broadens the scope of the CRISPR-Cas sensor for the detection of non-nucleic acid hazards in the environment and food samples.

A universal, portable, and ultra-sensitive pipet immunoassay platform for deoxynivalenol detection based on dopamine self-polymerization-mediated bioconjugation and signal amplification.

Deoxynivalenol (DON) is highly toxic to the environment and human health. It is important to detect DON with ultra-high sensitivity, ease of operation, and low cost. Inspired by the excellent stability and biocompatibility of polydopamine, a universal, portable and ultra-sensitive pipet immunoassay platform was reported for DON detection based on dopamine self-polymerization (polydopamine coating and polydopamine nanoparticles). The polydopamine coating acted as an effective strategy for biomolecule immobilization on the pipet to improve the coating efficiency that significantly reduced the required concentration of biomolecules. Performing the ELISA in pipets saved nearly 67% of the antigen amount and 83% of the antibody amount, which reduced the detection cost and simplified the experimental steps. The dual signal amplification in this pipet immunoassay enabled ultra-high sensitivity. Polydopamine nanoparticles acted as the enrichment carrier of horseradish peroxidase-goat anti-mouse IgG for the first-round signal amplification, followed by the tyramine-mediated loading of streptavidin-HRP for the second-round signal amplification. The dual-enriched HRP catalyzed the color-developing substrate to achieve highly sensitive colorimetric DON detection with a limit of detection of 0.435 ng/mL.