Quantum-Dot-Based Enzyme Biosensor for Rapid Screening of Antibiotic Susceptibility in Urine Samples

The rising threat of antibiotic resistance necessitates sensitive and specific diagnostic tools for antimicrobial susceptibility testing (AST). Gram-negative bacteria are catalase-positive and the major group that causes urinary tract infections (UTI). The present work focused on developing a novel quantum dot (QD) based dual enzyme assay to differentiate antibiotic-resistant and antibiotic-sensitive bacteria within 2 h. The first step involves oxidation of dopamine on the surface of QDs by a horseradish peroxidase (HRP) enzyme in the presence of hydrogen peroxide (H₂O₂), forming quinone, which quenches the fluorescence of QDs. However, in the presence of the catalase enzyme, H₂O₂ is catabolized, preventing dopamine oxidation and restoring the QD fluorescence. When treated with antibiotics, resistant bacteria produce catalase; in contrast, sensitive bacteria, with little or no catalase production, modulate QD fluorescence. The QD fluorescence was quenched by the HRP-Dopa system, <i>F</i>/<i>F</i> _o 0.63-0.03, whereas the presence of catalase led to fluorescence increase with <i>F</i>/<i>F</i> _o 3.53-9.8. Upon treating bacteria with various antibiotics, the resistant strains led to fluorescence enhancement with <i>F</i>/<i>F</i> _o ∼ 2-3, whereas sensitive strains led to <i>F</i>/<i>F</i> _o ∼ 1-1.2 with <i>P</i>-values of <i>P</i> < 0.05. The assay demonstrated a clinically relevant detection threshold of 10⁴-10⁵ CFU/mL for bacteriuria, affirming its relevance in early-stage infection detection and enhancing its diagnostic value. The developed biosensor achieved 100% specificity and was validated using urine samples. The biosensor distinguished resistant and sensitive strains within 2 h, offering a promising platform for rapid screening of antimicrobial susceptibility.