Magnetism-Resolved Separation and Fluorescence Quantification for Near-Simultaneous Detection of Multiple Pathogens

In the modern era of molecular evidence-based medicine and advanced biomedical technologies, the rapid, sensitive and specific assay of multiple pathogens is critical to, but largely absent from, clinical practice. Therefore, to improve the current ordinary separation and collection method, we report herein a strategy of magnetism-resolved separation and fluorescence quantification for near-simultaneous detection of multiple pathogens, followed by the direct antimicrobial susceptibility testing (AST). To accomplish this strategy, we utilized aptamer-modified fluorescent-magnetic multifunctional nanoprobes (apt-FMNPs). FMNPs with intriguing different magnetic responses and excellent fluorescence quality were first self-assembled based on metal coordination interaction using (3-mercaptopropyl) trimethoxysilane, magnetic γ-Fe₂O₃, and fluorescent quantum dots as matrix components. Then, aptamers, which specific to target pathogens of <i>Escherichia coli O157:H7</i> (<i>E. coli</i>) and <i>Salmonella typhimurium</i> (<i>S. typ</i>), were conjugated with FMNPs to yield apt-FMNPs nanoprobes for multiple pathogens assay. Based on the discrepant magnetic response of pathogen@nanoprobes complex under the identical external magnetic field, the model bacteria were fished out by magnetic adsorption at different time points and subjected to fluorescence quantification with good linear ranges and detection limits within 1h. Multiple pathogens spiked in real samples were also effectively detected by the apt-FMNPs and sequentially fished out for AST assay, which showed similar results to that for pure pathogens. The apt-FMNPs-based strategy of near-simultaneous detection of multiple pathogens shows promise for the potential application in the diagnosis and treatment of pathogen-related infectious diseases

Dual-Recognition Förster Resonance Energy Transfer Based Platform for One-Step Sensitive Detection of Pathogenic Bacteria Using Fluorescent Vancomycin–Gold Nanoclusters and Aptamer–Gold Nanoparticles

The effective monitoring, identification, and quantification of pathogenic bacteria is essential for addressing serious public health issues. In this study, we present a universal and facile one-step strategy for sensitive and selective detection of pathogenic bacteria using a dual-molecular affinity-based Förster (fluorescence) resonance energy transfer (FRET) platform based on the recognition of bacterial cell walls by antibiotic and aptamer molecules, respectively. As a proof of concept, Vancomycin (Van) and a nucleic acid aptamer were employed in a model dual-recognition scheme for detecting Staphylococcus aureus (Staph. aureus). Within 30 min, by using Van-functionalized gold nanoclusters and aptamer-modified gold nanoparticles as the energy donor and acceptor, respectively, the FRET signal shows a linear variation with the concentration of Staph. aureus in the range from 20 to 10⁸ cfu/mL with a detection limit of 10 cfu/mL. Other nontarget bacteria showed negative results, demonstrating the good specificity of the approach. When employed to assay Staph. aureus in real samples, the dual-recognition FRET strategy showed recoveries from 99.00% to the 109.75% with relative standard derivations (RSDs) less than 4%. This establishes a universal detection platform for sensitive, specific, and simple pathogenic bacteria detection, which could have great impact in the fields of food/public safety monitoring and infectious disease diagnosis