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.

"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.

Contamination-free V-shaped ultrafast reaction cascade transferase signal amplification driven CRISPR/Cas12a magnetic relaxation switching biosensor for bacteria detection.

Foodborne pathogenic bacteria seriously endanger human health and must be rapidly identified for control. Magnetic relaxation switching biosensors (MRS) are ideal for rapid bacteria detection due to their high signal-to-noise ratio and immunity to sample matrix signal interference. However, conventional MRS still has some challenges in terms of sensitivity, specificity, and stability due to insufficient cross-linking or non-specific binding of magnetic nanoparticles (MNPs) to the target. To address these challenges, we firstly proposed a novel contamination-free uracil-DNA glycosylase (UDG) assisted V-shaped PCR driven CRISPR/Cas12a-MRS (UPC-MRS) biosensor, which combines contamination-free ultrafast nucleic acid amplification and powerful CRISPR/Cas12a system. It has an extremely specific quadruple signal guarantee realized by the merits of UDG anti-contamination, PCR primer specificity matching, the CRISPR/Cas12a system's precise recognition abilities, and magnetic probe signal unaffected by the sample matrix. As a cascade combined with original terminal deoxynucleotidyl transferase (Tdt)-mediated signal amplification technology, this platform can achieve Salmonella detection at concentrations as low as 53 CFU/mL, which is more sensitive than most existing MRS sensors, and it displays accuracy and applicability in real sample detection. This novel UPC-MRS biosensors avoid the common aerosol pollution problem of previous CRISPR/Cas12a systems which after combining with nucleic acid amplification, hence not only offers an alternative toolbox for Salmonella and other pathogen detection with satisfactory specificity and sensitivity, but also has potential for future applications across diverse fields.

"Three-in-one" Zr-MOF Multifunctional Carrier-mediated Fluorescent and Colorimetric Dual-signal Readout Biosensing Platform to Enhance Analytical Performance.

To address the urgent demand for sensitive and stable detection applications, significant efforts have been made in the development of dual-signal readout assays for precise target detection and timely health risk control. Here, a new nanomaterial, Pt@PCN-224-HRP-initiator DNA (PP-HRP-iDNA), was exploited to construct a dual-signal readout biosensing platform. Zr-MOF (PCN-224) was loaded with as many Pt nanoparticles (NPs) and as much horseradish peroxidase (HRP) as possible to enhance the brightness of the colorimetric signal recognizable to the naked eye while also acting as a gatekeeper to protect the enzyme activity and ensuring the stability of the assay process. Moreover, the Pt NPs and HRP displayed a synergistic catalytic effect, which promoted the sensitivity of detection. Further, the formation of the Zr-O-P bond eliminated the instability of the interactions between PCN-224 and iDNA in a controllable manner. After the immunoreaction, iDNA stimulated a hybridization chain reaction, resulting in a significant reduction of the fluorescent DNA in the supernatant and a fluorescent signal change. Subsequently, the PP-HRP-iDNA probe implemented UV-light response (450 nm) where 3,3',5,5'-tetramethylbenzidine was used as a substrate for the colorimetric signal readout. By virtue of the nanomaterial-modulated transduction mechanism and the antigen-antibody interactions, this dual-signal biosensor displays high sensitivity, with a limit of detection of 0.65 pg/mL for aflatoxin B1 and 4 CFU/mL for Salmonella enteritidis, suggesting the detection potential of the biosensing platform for analyzing various targets.

Enzyme-free catalytic hairpin assembly reaction-mediated micro-orifice resistance assay for the ultrasensitive and low-cost detection of Listeria monocytogenes.

The rapid, reliable, ultra-sensitive, and cost-saving detection of Listeria monocytogenes (L. monocytogenes) has substantial implications for food safety. Thus, we developed a novel, enzyme-free, dual-signal amplification approach to detect L. monocytogenes based on the micro-orifice resistance technique combined with the aggregation of polystyrene (PS) microspheres constructed by the catalytic hairpin assembly reaction (CHA). Both the detection probes (probe2) and trigger DNA (tDNA) were first modified on PS microspheres (probe2-PS-tDNA). The tDNA was enriched by PS microspheres for the first signal amplification. After the hybridization reaction (the capture probes (probe1), target DNA, and probe2), unreacted probe2-PS-tDNA was removed, and the complex triggered the CHA reaction for the second signal amplification. Additionally, the micro-orifice resistance technique can sensitively identify PS microsphere aggregation caused by the CHA reaction to analyze the target DNA quantitatively. The CHA-mediated micro-orifice resistance assay was constructed by combining cost-saving PS microsphere probes, the highly specific DNA hybridization reaction, and the enzyme-free signal amplification strategy, substantially reducing the cost and improving the detection sensitivity (the limit of detection is 4 CFU/mL). This study provides a superior means to detect L. monocytogenes in complex food samples.

One-step homogeneous micro-orifice resistance immunoassay for detection of chlorpyrifos in orange samples.

In this work, a one-step homogeneous micro-orifice resistance immunoassay has been proposed for chlorpyrifos detection by integrating functionalized polystyrene (PS) microsphere probes with particle counting technology. The particle counter is highly sensitive and accurate for detecting the state of PS microspheres, where the particles of different states exhibit significant differences in resistance. The state of the functionalized PS microspheres is altered from dispersed to aggregated during the antigen-antibody recognition. Based on the degree of aggregation of the functionalized PS microsphere probes, chlorpyrifos can be quantitatively detected through the competitive immune response between PS antibodies and PS complete antigens. This one-step homogeneous micro-orifice resistance immunoassay simplified the procedures and greatly increased the sensitivity of detection, which has been successfully applied to detect chlorpyrifos in orange samples within 0.5 h, with the detection limit of 0.058 ng/mL.