Click Reaction-Mediated Fluorescent Immunosensor Based on Cu-MOF Nanoparticles for Ultrasensitive and High-Throughput Detection of Aflatoxin B1 in Food Samples.

Due to the high toxicity of aflatoxin B1 and its risks to human health, we developed a click reaction-mediated automated fluorescent immunosensor (CAFI) for sensitive detection of aflatoxin B1 based on the Cu(I)-catalyzed click reaction. With its large specific surface area, a copper-based metal-organic framework (Cu-MOF) was synthesized to adsorb and enrich the copper ion (Cu(II)) and then load the complete antigen (BSA-AFB1). After the immunoreaction, Cu(II) inside the Cu-MOF-Antigen conjugate would be reduced to Cu(I) in the presence of sodium ascorbate, which triggered the click reaction between the fluorescent donor-modified DNA and the receptor-modified complementary DNA to lead to a fluorescence signal readout. The whole reaction steps were finished by the self-developed automated immunoreaction device. This CAFI method showed a limit of detection (LOD) of 0.48 pg/mL as well as a 670-fold enhancement in sensitivity compared to conventional ELISA, revealing its great potential in practical applications and automated detection.

Bimetallic nanozyme-bioenzyme hybrid material-mediated ultrasensitive and automatic immunoassay for the detection of aflatoxin B1 in food.

Aflatoxin B1 (AFB1) is one of the most prevalent and dangerous biotoxin in crops and feedstuff, which poses a great threat to human health and also cause significant financial losses. Therefore, there is an urgent need to develop an effective method for AFB1 detection. In this work, we developed an automatic reaction equipment and nanozyme-enhanced immunosorbent assay (Auto-NEISA) for sensitive and accurate detection of AFB1 by combining the highly effective signal probes with a self-designed automated immunoreactive equipment. Wherein, polystyrene (PS) nanoparticles were used as signal carriers for loading a massive in situ-synthesized platinum and palladium bimetallic nanozyme, which could enrich horseradish peroxidase-labeled goat anti-mouse antibody (HRP-Ab2) on the nanozyme surface to form a bimetallic nanozyme-bioenzyme hybrid material for multiple signal amplification. The entire reaction could be automatically completed by the self-developed immunoreactive equipment. The Auto-NEISA method realized the sensitive detection of AFB1 with a wide linear detection range of 10-104 pg/mL, at a low limit of detection (LOD) of 5.52 pg/mL. The LOD was 65-fold lower than that of the enzyme-linked immunosorbent assay (ELISA). Additionally, Auto-NEISA was successfully applied to detect AFB1 in real food samples, demonstrating that it has considerable potential for detecting food contaminants with high accuracy and efficiency.

"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.

Polystyrene microsphere-mediated optical sensing strategy for ultrasensitive determination of aflatoxin M1 in milk.

Aflatoxin M1 (AFM1) contamination poses a serious threat to human health globally. Hence, it is necessary to develop reliable and ultrasensitive methods for the determination of AFM1 residue in food products at low levels. In this study, a novel polystyrene microsphere-mediated optical sensing (PSM-OS) strategy was constructed to solve the problems of low sensitivity and susceptibility to interference from the matrix in AFM1 determination. Polystyrene (PS) microspheres have the advantages of low cost, high stability, and controllable particle size. They can be useful optical signal probes for qualitative and quantitative analyses attributed to the fact that they have strong ultraviolet-visible (UV-vis) characteristic absorption peaks. Briefly, magnetic nanoparticles were modified with the complex of bovine serum protein and AFM1 (MNP150-BSA-AFM1), and biotinylated antibodies of AFM1 (AFM1-Ab-Bio). Meanwhile, PS microspheres were also functionalized with streptavidin (SA-PS950). In the presence of AFM1, a competitive immune reaction was triggered leading to the changes in AFM1-Ab-Bio concentrations on the surface of MNP150-BSA-AFM1. The complex of MNP150-BSA-AFM1-Ab-Bio binds with SA-PS950 to form the immune complexes due to the special binding of biotin and streptavidin. The remaining SA-PS950 in the supernatant was determined by UV-Vis spectrophotometer after magnetic separation, which positively correlated with the concentration of AFM1. This strategy allows for ultrasensitive determination of AFM1 with limits of detection as low as 3.2 pg/mL. It was also successfully validated for AFM1 determination in milk samples, and a high consistency was found with the chemiluminescence immunoassay. Overall, the proposed PSM-OS strategy can be used for the rapid, ultrasensitive, and convenient determination of AFM1, as well as other biochemical analytes.