A gelatinase-responsive cascade microfluidic chemiluminescence assay for ultrasensitive detection of drug-resistant bacteria and antibiotic susceptibility.

Antibiotic-resistant bacterial infections pose a global health crisis, driving the need for innovative diagnostic approaches. Conventional methods for detecting resistant strains and antibiotic susceptibility testing (AST) are often limited by long assay times, low sensitivity, and operational complexity. Herein, a novel gelatinase-responsive cascade microfluidic chemiluminescence (CL) biosensor was developed for ultrasensitive detection of gelatinase-producing bacteria, with phenotypic AST validated using Staphylococcus aureus (S. aureus) as the model pathogen. Peptide-DNA probes were designed to respond to gelatinase secreted by bacteria remain metabolically active following antibiotic exposure. Bacterial gelatinase cleaves peptides to release initiator single-stranded DNA (ssDNA), triggering hybridization chain reaction (HCR) amplification in micropillar-structured microchannels. Through streptavidin-biotin interaction, poly-horseradish peroxidase (poly-HRP) is anchored in the channel to catalyze luminol based CL. This biosensor achieves high specificity and sensitivity, with a detection limit of 15 CFU/mL, which is better or comparable to previously reported biosensors. Moreover, the potential clinical applicability of this platform is further verified by testing four clinical S. aureus strains against five antibiotics, yielding 95% categorical agreement rate with standard AST methods. The area under the receiver operating characteristic ROC curve (AUC) was 0.944, close to the ideal value of 1, indicating excellent diagnostic performance for AST. The assay time from isolated colonies to AST result takes approximately 4-5 h. Featuring portability and low cost, the platform enables universal bacterial AST, providing a reliable tool for precise antimicrobial therapy and antimicrobial resistance control.