Engineering of Dual-Recognition Ratiometric Fluorescent Nanosensor with Remarkable Large Stokes Shift for Accurate Tracking of Pathogenic Bacteria at Single-Cell Level.

Shen, Yizhong; Wu, Tingting; Zhang, Yiyin; Ling, Na; Zheng, Libing; Zhang, Shao-Lin; Sun, Yidan; Wang, Xiaohong; Ye, Yingwang

Rapid, accurate, reliable and risk-free tracking of pathogenic microorganism at single-cell level is critical to achieve efficient source control and prevent outbreaks of microbiological infectious diseases. In this work, we first reported a promising potential of integrating the concepts of remarkable large Stokes shift and dual-recognition into a single matrix for developing pathogenic microorganism stimuli-responsive ratiometric fluorescent nanoprobe with speediness, cost-efficiency, stability, ultrahigh specificity and sensitivity. As a proof-of-concept, we selected Gram-positive bacterium Staph-ylococcus aureus (S. aureus) as the targeting analyte model, which easily bound to its recognition aptamer and broad-spectrum glycopeptide antibiotic vancomycin (Van). To improve the specificity and short sample-to-answer time, we em-ployed the classic noncovalent - stacking interactions as a driving force to trigger Van and aptamer dual-functionalized near-infrared (NIR) fluorescence Apt-Van-QDs to bind to the surface of an unreported blue fluorescence of -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) got close to occur fluorescence resonance energy transfer (FRET) process, leading to a remarkable blue fluorescence quenching for CNPs at ~ 465 nm and a clear NIR fluorescence enhancement for Apt-Van-QDs at ~ 725 nm. In the presence of S. aureus, the FRET process from CNPs to Apt-Van-QDs was disrupted, rendering nanoprobe Apt-Van-QDs@CNPs to display a ratiometric fluo-rescent response to S. aureus, which exhibited ~ 260 nm large Stokes shift and rapid sample-to-answer detection time (~ 30.0 min). As expected, the nanoprobe Apt-Van-QDs@CNPs showed an ultrahigh specificity for ratiometric fluorescent de-tection of S. aureus with a promising 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 can give a new insight into the rational design of efficient ratiometric nanoprobes for rapidly on-site accurate screening of pathogenic microorganism at single-cell level in the early stages, especially during the worldwide spread of COVID-19 today.