Rapid, accurate, reliable, and risk-free tracking of pathogenic microorganisms at the single-cell level is critical to achieve efficient source control and prevent outbreaks of microbial infectious diseases. For the first time, we report a promising approach for integrating the concepts of a remarkably large Stokes shift and dual-recognition into a single matrix to develop a pathogenic microorganism stimuli-responsive ratiometric fluorescent nanoprobe with speed, cost efficiency, stability, ultrahigh specificity, and sensitivity. As a proof-of-concept, we selected the Gram-positive bacterium Staphylococcus aureus (S. aureus) as the target analyte model, which easily bound to its recognition aptamer and the broad-spectrum glycopeptide antibiotic vancomycin (Van). To improve the specificity and short sample-to-answer time, we employed classic noncovalent &#960;-&#960; stacking interactions as a driving force to trigger the binding of Van and aptamer dual-functionalized near-infrared (NIR) fluorescent Apt-Van-QDs to the surface of an unreported blue fluorescent &#960;-rich electronic carbon nanoparticles (CNPs), achieving S. aureus stimuli-responsive ratiometric nanoprobe Apt-Van-QDs@CNPs. In the assembly of Apt-Van-QDs@CNPs, the blue CNPs (energy donor) and NIR Apt-Van-QDs (energy acceptor) became close to allow the fluorescence resonance energy transfer (FRET) process, leading to a remarkable blue fluorescence quenching for the CNPs at â¼465 nm and a clear NIR fluorescence enhancement for Apt-Van-QDs at â¼725 nm. In the presence of S. aureus, the FRET process from CNPs to Apt-Van-QDs was disrupted, causing the nanoprobe Apt-Van-QDs@CNPs to display a ratiometric fluorescent response to S. aureus, which exhibited a large Stokes shift of â¼260 nm and rapid sample-to-answer detection time (â¼30.0 min). As expected, the nanoprobe Apt-Van-QDs@CNPs showed an ultrahigh specificity for ratiometric fluorescence detection of S. aureus with a good detection limit of 1.0 CFU/mL, allowing the assay at single-cell level. Moreover, we also carried out the precise analysis of S. aureus in actual samples with acceptable results. We believe that this work offers new insight into the rational design of efficient ratiometric nanoprobes for rapid on-site accurate screening of pathogenic microorganisms at the single-cell level in the early stages, especially during the worldwide spread of COVID-19 today.
Engineering of a Dual-Recognition Ratiometric Fluorescent Nanosensor with a Remarkably Large Stokes Shift for Accurate Tracking of Pathogenic Bacteria at the Single-Cell Level