A Dot-Array Magnetic Enrichment Strategy for Rapid, Multiplexed Detection of Viable Bacteria via ATP Bioluminescence

Abstract

The sensitive detection of microorganisms in complex samples, such as herbal medicines, was significantly hampered by severe matrix interference and low target abundance. While magnetic separation and microfluidics offered enrichment capabilities, existing platforms often struggled to achieve efficient, parallel enrichment of both specific pathogens and total aerobic microbes. To address this critical gap, we developed an integrated microfluidic platform featuring a custom dotarray magnetic field that enabled highly efficient and parallel enrichment of multiple bacterial targets. A dual-mechanism capture strategy was implemented, utilizing immunomagnetic beads (IMBs) for the specific isolation of Salmonella Paratyphi B (S. paratyphi B) and Escherichia coli (E. coil), and cationic polymer-modified beads for the broad-spectrum capture of aerobic bacteria. Following enrichment, captured viable bacteria were rapidly quantified via ATP bioluminescence. The system demonstrated high sensitivity, with limits of detection of 49 CFU/mL for S. Paratyphi B, 60 CFU/mL for E. coli, and 55 CFU/mL for aerobic bacteria, and exhibited a strong linear response (R² > 0.99) across a wide dynamic range (10³-10⁷ CFU/mL). Successful application in spiked licorice samples confirmed the platform's practicality and reliability for the rapid, multiplexed detection of microbial contaminants in complex herbal products, offering a powerful tool for quality and safety monitoring.