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CRISPR/Cas-based biosensors were typically used for nucleic-acid targets detection and complex DNA extraction and amplification procedures were usually inevitable. Here, we report a CRISPR/Cas12a-mediated, DNA extraction and amplification-free, highly direct and rapid biosensor (abbreviated as "CATCHER") for Salmonella Typhimurium (S. Typhimurium) with a simple (3 steps) and fast (∼2 h) sensing workflow. Magnetic nanoparticle immobilized anti-S. Typhimurium antibody was worked as capture probe to capture the target and provide movable reaction interface. Colloidal gold labeled with anti-S. Typhimurium antibody and DNase I was used as detection probe to bridge the input target and output signal. First, in the presence of S. Typhimurium, an immuno-sandwich structure was formed. Second, DNase I in sandwich structure degraded the valid, complete activator DNA to invalid DNA fragments which can't trigger the trans-cleavage activity of Cas12a. Finally, the integrity of reporter DNA was preserved presenting a low fluorescence signal. Conversely, in the absence of S. Typhimurium, strong fluorescence recovery appeared owing to the cutting of reporter by activated Cas12a. Significantly, the proposed "CATCHER" showed satisfactory detection performance for S. Typhimurium with the limit of detection (LOD) of 7.9 × 10

Salmonella is a common pathogenic bacterium. Salmonella isolates have been sorted into different serogroups according to the specific lipopolysaccharide present in the somatic surface (O antigen) of the bacterium. The virulence and pathogenicity of Salmonella vary greatly between different serogroups. Salmonella of O:2, O:3, O:4, O:7, and O:9 serogroups contribute to a large share of Salmonella outbreaks. Traditional slide agglutination tests to classify Salmonella serogroups are tedious because of their low testing efficiency, complex operation, and subjective judging criteria. Immunochromatographic assays have been extensively described in the literature, which discussed their low cost, rapid detection, and portability in multitude of fields, such as agriculture and environmental sciences. In this study, a biosensing multiplexer based on immunochromatographic assay (bio-MUX) was designed for rapid and high-throughput classification of five Salmonella serogroups. The five tests for the serogroups (i.e., O:2, O:3, O:4, O:7, and O:9) were integrated in three test lines. The results can be exported rapidly in 15 min. The sensitivity and specificity for the five serogroups exceeded 96% and 97%, respectively. No expensive or large instruments were needed in the bio-MUX. With these advantages, the proposed bio-MUX deserves to be promoted and expanded for on-site circumstances and specialized laboratories.

Pathogens, mycotoxins, or antibiotics may exist in a food sample. Micro- and macromolecular substances must be detected quickly. A rapid and convenient lateral flow immunoassay (LFI) integrated with competitive and sandwich models was developed to detect micro- and macromolecular substances. In this study, aflatoxin M1 (AFM1) and Escherichia coli O157:H7 were selected as the micro- and macromolecular substances, respectively. Two test lines in the LFI test strip were evaluated to detect AFM1 and E. coli O157:H7 by competitive and sandwich models. Results showed that the limits of detection for detecting AFM1 and E. coli O157:H7 were 50 pg·mL−1 and 1.58 × 104 cfu·mL−1, respectively. The whole assay time was 30 min. The recoveries of gold nanoparticle-LFI ranged from 78.0 to 111.6% with coefficients of variation in the range of 3.9 to 8.5% for the detection of AFM1. For the detection of E. coli O157:H7, the range of recoveries was from 70.1 to 89.6% with coefficients of variation ranging from 4.9 to 13.0%. This study not only tested sensitivity and specificity, but also was a systematic study of location of 2 test lines of the LFI test strip integrated with competitive and sandwich models.

A pre-concentrated immunochromatographic assay for Salmonella enteritidis (S. enteritidis) detection was developed based on the unique optical and magnetic properties of magnetic nanoparticles (MNPs). The target was easily concentrated from a large volume of sample by using immune MNPs (IMNPs) with superparamagnetism. The results could be directly distinguished using colored IMNPs on test strip. A target elution step was not required in the proposed method. The key parameters, including coupling pH, blocking agents, and amount of IMNPs on the test strip, were optimized. Colloidal gold-based immunochromatographic assay (CG-ICA), MNP-based immunochromatographic assay (MNP-ICA), and MNP-ICA for a large-volume sample (large-volume MNP-ICA) were compared. Under optimal conditions, the sensitivity of large-volume MNP-ICA was 1.95 × 105 CFU mL−1, which was 10 times higher than that of MNP-ICA and 1000 times higher than that of CG-ICA, owing to the pre-concentration from a large sample volume. Moreover, the antibody consumption of large-volume MNP-ICA was lower than that of conventional CG-ICA. The entire detection time of the proposed method was only 30 min. In general, the proposed method may be considered as a rapid, sensitive, and simple screening tool for on-site detection of S. enteritidis as well as other pathogens.

Various pathogens may coexist in one sample; however, detection methods that rely on traditional selective culture media or immune agents designed specifically for a certain target are unsuitable for multiple targets. It is important to develop a simultaneous and sensitive detection method for multiple pathogens. Here, a multicolor and ultrasensitive enzyme-linked immunosorbent assay (ELISA) platform based on the fluorescence hybridization chain reaction (HCR) was developed. In the assay, multicolor fluorescence concatemers formed as signal amplifiers and signal reporters in the presence of target pathogens. When HCR occurred, Escherichia coli O157:H7, Salmonella serotype Choleraesuis, and Listeria monocytogenes were detected simultaneously with three different fluorescences. Additionally, the limits of detection for E. coli O157:H7, Salmonella Choleraesuis, and L. monocytogenes were 3.4 × 101, 6.4 × 100, and 7.0 × 101 CFU/mL, respectively. The assay achieved ultrasensitive, specific, and simultaneous detection of three pathogens and can be applied to the detection of pathogens in milk samples. Therefore, this multicolor and ultrasensitive ELISA platform has great potential in the application of simultaneous detection of pathogens.

Escherichia coli O157:H7 makes a major threat to human health. Aiming to detect Escherichia coli O157:H7 sensitively, hybridization chain reaction signal amplified immunoassay (immuno-HCR) based on contact quenching (CQ) and fluorescence resonance energy transfer (FRET) was developed. The background of the new designed HCR hairpins (CQ-FRET hairpins) was reduced by contact-quenching fluorescein (FAM) and breaking FRET from donor (FAM) to acceptor (Cy5). The F/F0 ratio of CQ-FRET hairpins (37.02) was obviously higher than that of two other common HCR fluorescent hairpins (CQ hairpins, 21.45; FRET hairpins, 4.61). The limit of detection of the assay was 3.5 × 101 CFU/mL and obviously lower than that of CQ hairpins based immuno-HCR (3.28 × 103 CFU/mL) and FRET hairpins based immuno-HCR (6.49 × 104 CFU/mL). The proposed low fluorescent background immuno-HCR with high sensitivity which was verified in contaminated milk samples could be potentially used in the detection of various pathogens.

Fluorescent lateral flow immunoassay (FLFIA) based on immunomagnetic separation (IMS) has the advantage of sensitivity. However, its complex operation includes IMS, elution, incubation, and FLFIA steps. Here, we prepared a core@shell@satellite structure fluorescent magnetic nanobeads (FMNBs) and firstly introduced them into the novel method that integrated IMS with FLFIA (I-IMS-FLFIA) for the detection of Escherichia coli O157:H7. The FMNBs exhibited excellent magnetic and fluorescent properties for applications in IMS and FLFIA. However, the inner filter effect (IFE) of FMNBs may disturb the detection of I-IMS-FLFIA. Systematical studies showed that the amount of immuno-FMNBs and the concentration of monoclonal antibody can be controlled to obtain maximum photoluminescence intensity and effectively weaken or solve IFE. Under optimum conditions, this method allows for the quantified detection of 2.39 × 102 CFU/mL and qualified detection of 2.50 × 103 CFU/mL. The method is simple, safe, efficient, and sensitive for the detection of foodborne pathogens.