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

Abstract

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, F/Fo 0.63–0.03, whereas the presence of catalase led to fluorescence increase with F/Fo 3.53–9.8. Upon treating bacteria with various antibiotics, the resistant strains led to fluorescence enhancement with F/Fo ∼ 2–3, whereas sensitive strains led to F/Fo ∼ 1–1.2 with P-values of P < 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.