A Dual-Mode “Turn-On” Ratiometric Luminescent Sensor Based on Upconverting Nanoparticles for Detection and Differentiation of Gram-Positive and Gram-Negative Bacteria

Infectious bacterial diseases, intensified by antibiotic resistance, cause millions of deaths annually and pose risks beyond human health, including water and food contamination. Current diagnostics are often slow, require complex equipment, and lack specificity, highlighting the need for rapid and reliable detection methods. To address this, we developed a luminescent sensor based on NaYF₄ upconverting nanoparticles (UCNPs) doped with Er³⁺ or Tm³⁺, coated with COOH-PEG₄-COOH, and functionalized with vancomycin (Van) or polymyxin-B (Poly) to selectively target Gram-positive and Gram-negative bacteria, respectively. Gold nanoparticles (AuNPs) served as quenchers, enabling a ratiometric "turn-on" mechanism: upon bacterial binding, the UCNP emission, initially quenched by AuNPs, was partially restored. This allowed differentiation through changes in the green/red (G/R) ratio for Er-UCNP@PEG₄-Van and the blue/red (B/R) ratio for Tm-UCNP@PEG₄-Poly. The sensor distinguished between Gram-positive and Gram-negative bacteria over a wide concentration range (0.05 to 5 × 10⁵ CFU/mL) and showed high correlation with actual bacterial counts (<i>r</i> = 0.99 for <i>S. aureus</i>, <i>r</i> = 0.91 for <i>E. coli</i>). This platform is a potential fast, selective, and reliable tool for bacterial detection in clinical and environmental settings.