An Ultrasensitive Colorimetric Foodborne Pathogenic Detection Method Using a CRISPR/Cas12a Mediated Strand Displacement/Hybridization Chain Reaction.

Accurate, rapid, and sensitive pathogenic detections play an important role in food safety. Herein, we developed a novel CRISPR/Cas12a mediated strand displacement/hybridization chain reaction (CSDHCR) nucleic acid assay for foodborne pathogenic colorimetric detection. A biotinylated DNA toehold is coupled on avidin magnetic beads and acts as an initiator strand to trigger the SDHCR. The SDHCR amplification allowed the formation of long hemin/G-quadruplex-based DNAzyme products to catalyze the TMB-H2O2 reaction. In the presence of the DNA targets, the trans-cleavage activity of CRISPR/Cas12a was activated to cleave the initiator DNA, resulting in the failure of SDHCR and no color change. Under optimal conditions, the CSDHCR has a satisfactory linear detection of DNA targets with a regression equation Y = 0.0531*X - 0.0091 (R2 = 0.9903) in the range of 10 fM to 1 nM, and the limit of detection was determined as 4.54 fM. In addition, Vibrio vulnificus, one foodborne pathogen, was used to verify the practical application of the method, and it showed satisfactory specificity and sensitivity with a limit of detection at 1.0 × 100 CFU/mL coupling with recombinase polymerase amplification. Our proposed CSDHCR biosensor could be a promising alternative method for ultrasensitive and visual detection of nucleic acids and the practical application of foodborne pathogens.

One-step synthesized antimicrobial peptide-functionalized gold nanoclusters for selective imaging and killing of pathogenic bacteria.

The development of multifunctional nanomaterials with bacterial imaging and killing activities is of great importance for the rapid diagnosis and timely treatment of bacterial infections. Herein, peptide-functionalized gold nanoclusters (CWR11-AuNCs) with high-intensity red fluorescence were successfully synthesized via a one-step method using CWR11 as a template and by optimizing the ratio of CWR11 to HAuCl4, reaction time, pH, and temperature. The CWR11-AuNCs bound to bacteria and exhibited selective fluorescence microscopy imaging properties, which is expected to provide a feasible method for locating and imaging bacteria in complex in vivo environments. In addition, CWR11-AuNCs not only retained the antibacterial and bactericidal activities of CWR11 but also exhibited certain inhibitory or killing effects on gram-negative and gram-positive bacteria and biofilms. The MICs of CWR11-AuNCs against Escherichia coli and Staphylococcus aureus were 178 and 89 µg/ml, respectively. Surprisingly, cell viability in the CWR11-AuNC-treated group was greater than that in the CWR11-treated group, and the low cytotoxicity exhibited by the CWR11-AuNCs make them more promising for clinical applications.

Cu-MOF@Pt 3D nanocomposites prepared by one-step wrapping method with peroxidase-like activity for colorimetric detection of glucose.

As an alternative to natural enzymes, artificial enzymes based on nanomaterials have attracted a lot of attention owing to their outstanding catalytic activity and high stability as well as low cost. Cu-MOF loaded with platinum nanoparticles (labeled Cu-MOF@Pt) was prepared by simple one-step wrapping method using platinum nanoparticles, copper nitrate trihydrate and 1,3,5-tricarboxybenzene. It was confirmed that Cu-MOF@Pt exhibits peroxidase-like activity, which can quickly catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) and produce blue oxidized TMB (oxTMB) in the presence of hydrogen peroxide (H2O2). Additionally, steady-state kinetics showed that Cu-MOF@Pt exhibits stronger appetency to TMB and H2O2 compared with horseradish peroxidase. Thanks to the peroxidase-like activity of Cu-MOF@Pt, a highly selective colorimetric method for glucose detection has been successfully established, the linear range is 2-15 mM (R2 =0.9999) and the Limit of Detection (LOD) is 0.42 mM, with a detection range that meets clinical needs. Moreover, its good intra- and inter-assay precision and excellent stability make the results of glucose detection very reproducible. The detection performance of 90.09% was still maintained at 4 â„ƒ for 2 months. In conclusion, a new nanocomposite was successfully prepared and its selective detection ability for glucose was proved, which established a good basis for the clinical development of new enzymes for biosensors.

Rapid photothermal detection of foodborne pathogens based on the aggregation of MPBA-AuNPs induced by MPBA using a thermometer as a readout.

Rapid and portable detection of foodborne pathogens is of great significance for food safety and public health. The colorimetric methods based on naked-eye have been demonstrated to be a suitable qualitative method for point-of-care testing (POCT). However, analytical instruments like a microplate reader must be needed for the quantitative assay. To overcome its limitation, we herein report a novel photothermal method for foodborne pathogens based on the photothermal effect of aggregated mercaptophenylboronic acid-functionalized AuNPs (MPBA-AuNPs) induced by MPBA to translate the colorimetric detection into a simple temperature measurement using thermometers as the readout. The aggregated AuNPs show higher photothermal conversion efficiency than well-separated AuNPs under 660 nm laser irradiation. In the presence of bacteria, MPBA-AuNPs will attach to the surface of bacteria and keep separated from aggregation induced by excess MPBA, resulting in a lower temperature increase under 660 nm laser irradiation. Using E. coli O157:H7 as a model target, a good linear relationship is observed between temperature increase and bacteria concentration from 1.00 × 105-1.00 × 109 cfu mL-1 (R2 = 0.9877) with a detection limit of 1.97 × 104 cfu mL-1, which is three orders of magnitude lower than of the MPBA-AuNPs-based colorimetric assays. The proposed photothermal method provided a universal platform for rapid and portable detection of broad-spectrum bacteria strains in real samples.

Rapid Detection and Antimicrobial Susceptibility Testing of Pathogens Using AgNPs-Invertase Complexes and the Personal Glucose Meter.

Rapid detection of pathogens and assessment of antimicrobial susceptibility is of great importance for public health, especially in resource-limiting regions. Herein, we developed a rapid, portable, and universal detection method for bacteria using AgNPs-invertase complexes and the personal glucose meter (PGM). In the presence of bacteria, the invertase could be released from AgNPs-invertase complexes where its enzyme activity of invertase was inhibited. Then, the enzyme activity of invertase was restored and could convert sucrose into glucose measured by a commercially PGM. There was a good linear relationship between PGM signal and concentration of E. coli or S. aureus as the bacteria model with high sensitivity. And our proposed biosensor was proved to be a rapid and reliable method for antimicrobial susceptibility testing within 4 h with consistent results of Minimum Inhibitory Concentrations (MICs) testing, providing a portable and convenient method to treat infected patients with correct antibiotics and reduce the production of antibiotic-resistant bacteria, especially for resource-limiting settings.